Composition and methods for treating cancer and immune disorders using veillonella bacteria

ABSTRACT

Provided herein are methods and compositions related to Veillonella bacteria useful as therapeutic agents.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/268,745, filed Feb. 6, 2019, which claims the benefit of priority toU.S. Provisional Patent Application Ser. Nos. 62/626,789, filed Feb. 6,2018, 62/666,944, filed May 4, 2018, and 62/703,269, filed Jul. 25,2018, the contents of each of which are hereby incorporated herein byreference in their entirety.

SUMMARY

In certain aspects, provided herein are methods and compositions (e.g.,bacterial compositions, pharmaceutical compositions) related to thetreatment and/or prevention of disease (e.g., cancer, autoimmunedisease, inflammatory disease, metabolic disease), in a subject (e.g., ahuman subject) comprising administering a bacterial compositioncomprising Veillonella bacteria (e.g., Veillonella tobetsuensis,Veillonella parvula) and/or a product of such bacteria (e.g.,extracellular vesicles (EVs) and/or pharmaceutically active biomasses(PhABs)). Also provided herein are methods of making and/or identifyingsuch a bacterium and/or bacterial product. In some embodiments, providedhere are bioreactors comprising such bacteria. In some embodiments, theVeillonella bacteria (e.g., Veillonella tobetsuensis, Veillonellaparvula) is a strain of bacteria listed in Table 1. In some embodiments,the bacteria is a strain comprising at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% sequence identity (e.g., at least99.5% sequence identity, at least 99.6% sequence identity, at least99.7% sequence identity, at least 99.8% sequence identity, at least99.9% sequence identity) to the nucleotide sequence (e.g., genomicsequence, 16S sequence, CRISPR sequence) of the bacterial strains listedin Table 1. In some embodiments, the administration of the bacterialcomposition treats the immune disorder in the subject. In someembodiments, the immune disorder is an autoimmune disease. In someembodiments, the immune disorder is an inflammatory disease. In someembodiments, the immune disorder is an allergy.

In some embodiments, provided herein are extracellular vesicles (EVs)produced by and/or generated by and/or isolated from Veillonellabacteria (e.g., Veillonella tobetsuensis, Veillonella parvula) providedherein. In some embodiments, the bacterial compositions comprise bothVeillonella EVs and whole Veillonella bacteria (e.g., live bacteria,killed bacteria, attenuated bacteria). In certain embodiments, providedherein are bacterial compositions comprising Veillonella bacteria in theabsence of Veillonella EVs. In some embodiments, the pharmaceuticalcompositions comprise Veillonella EVs in the absence of Veillonellabacteria.

In certain embodiments, provided herein are methods of treating asubject who has an immune disorder (e.g., an autoimmune disease, aninflammatory disease, an allergy), comprising administering to thesubject a bacterial composition comprising a Veillonella bacterium(e.g., a killed bacterium, a live bacterium and/or an attenuatedbacterium). In certain embodiments, provided herein are methods oftreating a subject who has a metabolic disease comprising administeringto the subject a bacterial composition described herein. In someembodiments, the bacterium is a strain of bacteria listed in Table 1. Insome embodiments, the bacterium is a strain comprising at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% sequence identity(e.g., genomic sequence identity, 16S sequence identity, CRISPR sequenceidentity) (e.g., at least 99.5% sequence identity, at least 99.6%sequence identity, at least 99.7% sequence identity, at least 99.8%sequence identity, at least 99.9% sequence identity) to thecorresponding nucleotide sequence of the bacterial strains listed inTable 1. In some embodiments, at least 50%, 60%, 70%, 80%, 85%, 90%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the bacteria inthe bacterial composition are bacterial strains listed in Table 1. Insome embodiments, all or substantially all of the bacteria in thebacterial formulation are bacterial strains listed in Table 1. In someembodiments, the bacterial formulation comprises at least 1×10⁵, 5×10⁵,1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷,2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸,3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸ or 1×10⁹ colony formingunits of Veillonella bacteria (e.g., a strain of bacteria listed inTable 1).

In certain embodiments, provided herein are methods of treating asubject who has cancer comprising administering to the subject abacterial composition described herein.

In some embodiments, the method further comprises administering to thesubject an antibiotic. In some embodiments, the method further comprisesadministering to the subject one or more other cancer therapies (e.g.,surgical removal of a tumor, the administration of a chemotherapeuticagent, the administration of radiation therapy, and/or theadministration of a cancer immunotherapy, such as an immune checkpointinhibitor, a cancer-specific antibody, a cancer vaccine, a primedantigen presenting cell, a cancer-specific T cell, a cancer-specificchimeric antigen receptor (CAR) T cell, an immune activating protein,and/or an adjuvant).

In some embodiments, the method further comprises the administration ofanother therapeutic bacterium. In some embodiments, the method furthercomprises the administration of an immune suppressant and/or ananti-inflammatory agent. In some embodiments, the method furthercomprises the administration of a metabolic disease therapeutic agent.

In certain embodiments, provided herein are bacterial compositionscomprising a bacterial strain listed in Table 1 (e.g., a killedbacterium, a live bacterium and/or an attenuated bacterium) and/or aproduct of such bacteria (e.g., extracellular vesicles (EVs) and/orpharmaceutically active biomasses (PhABs)). In some embodiments, atleast 50%, 60%, 70%, 80%, 85%, 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% of the bacteria in the bacterial composition are astrain of bacteria listed in Table 1. In some embodiments, the bacteriais a strain of bacteria listed in Table 1. In some embodiments, thebacteria is a strain comprising at least 99% sequence identity (e.g.,genomic sequence identity, 16S sequence identity, CRISPR sequenceidentity) (e.g., at least 99.5% sequence identity, at least 99.6%sequence identity, at least 99.7% sequence identity, at least 99.8%sequence identity, at least 99.9% sequence identity) to the nucleotidesequence of the strain of bacteria listed in Table 1. In someembodiments, all or substantially all of the bacteria in the bacterialformulation are a bacterial strain listed in Table 1. In someembodiments, the bacterial formulation comprises at least 1×10⁵, 5×10⁵,1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷,2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸,3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸ or 1×10⁹ colony formingunits of a bacterial strain listed in Table 1. In some embodiments, thebacterial composition comprises EVs and/or PhABs (e.g., whole cells,fractions of cells, supernatant from fermentation, fractions ofsupernatant and/or extracellular vesicles) made from a bacterial strainlisted in Table 1.

In some embodiments, the bacterial composition is administered orally,intravenously, intratumorally, or subcutaneously. In some embodiments,the bacterial composition is administered in 2 or more (e.g., 3 or more,4 or more or 5 or more doses). In some embodiments, the administrationto the subject of the two or more doses are separated by at least 1hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16hours, 17 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or 21 days.In some embodiments, a second bacterium is administered as part of anecological consortium.

In certain embodiments, the composition comprises a specific ratio ofVeillonella bacteria to Veillonella EV particles. For example, in someembodiments, the pharmaceutical composition comprises at least 1Veillonella bacterium for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2,6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18.19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29, 30, 31, 32, 33, 34, 35, 36,37, 38. 39, 40, 41, 42, 43, 44, 45, 46, 47, 48. 49, 50, 51, 52, 53, 54,55, 56, 57, 58. 59, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 72,73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88. 89, 90,91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1×10³, 2×10³,3×10³, 4×10³, 5×10³, 6×10³, 7×10³, 8×10³, 9×10³, 1×10⁴, 2×10⁴, 3×10⁴,4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵,5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶,6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷,7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸,8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹,9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰,9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹,9×10¹¹, and/or 1×10¹² Veillonella EV particles. In some embodiments, thepharmaceutical composition comprises about 1 Veillonella bacterium forevery 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3,2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8,3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3,5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8,6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3,8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8,9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, 20, 21, 22, 23, 24, 25, 26,27, 28. 29, 30, 31, 32, 33, 34, 35, 36, 37, 38. 39, 40, 41, 42, 43, 44,45, 46, 47, 48. 49, 50, 51, 52, 53, 54, 55, 56, 57, 58. 59, 60, 61, 62,63, 64, 65, 66, 67, 68. 69, 70, 71, 72, 73, 74, 75, 76, 77, 78. 79, 80,81, 82, 83, 84, 85, 86, 87, 88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98.99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1×10³, 2×10³, 3×10³, 4×10³, 5×10³, 6×10³,7×10³, 8×10³, 9×10³, 1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴,8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵,9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶,1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸,2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹,3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰,4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹,4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, and/or 1×10¹²Veillonella EV particles. In some embodiments, the pharmaceuticalcomposition comprises no more than 1 Veillonella bacterium for every 1,1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5,2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4,4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5,5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7,7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5,8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10,11, 12, 13, 14, 15, 16, 17, 18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28.29, 30, 31, 32, 33, 34, 35, 36, 37, 38. 39, 40, 41, 42, 43, 44, 45, 46,47, 48. 49, 50, 51, 52, 53, 54, 55, 56, 57, 58. 59, 60, 61, 62, 63, 64,65, 66, 67, 68. 69, 70, 71, 72, 73, 74, 75, 76, 77, 78. 79, 80, 81, 82,83, 84, 85, 86, 87, 88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,850, 900, 950, 1×10³, 2×10³, 3×10³, 4×10³, 5×10³, 6×10³, 7×10³, 8×10³,9×10³, 1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴,1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶,2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷,3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸,4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹,5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰,5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹,5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, and/or 1×10¹² Veillonella EVparticles. In some embodiments, the pharmaceutical composition comprisesat least 1 Veillonella EV particle for every 1, 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3,3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5,4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6,6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5,7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9,9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16,17, 18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29, 30, 31, 32, 33, 34,35, 36, 37, 38. 39, 40, 41, 42, 43, 44, 45, 46, 47, 48. 49, 50, 51, 52,53, 54, 55, 56, 57, 58. 59, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70,71, 72, 73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88.89, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1×10³,2×10³, 3×10³, 4×10³, 5×10³, 6×10³, 7×10³, 8×10³, 9×10³, 1×10⁴, 2×10⁴,3×10⁴, 4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵, 3×10⁵,4×10⁵, 5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶,5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷,6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸,7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹,8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰,8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹,8×10¹¹, 9×10¹¹, and/or 1×10¹² Veillonella bacterium. In someembodiments, the pharmaceutical composition comprises about 1Veillonella EV particle for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2,6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18.19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29, 30, 31, 32, 33, 34, 35, 36,37, 38. 39, 40, 41, 42, 43, 44, 45, 46, 47, 48. 49, 50, 51, 52, 53, 54,55, 56, 57, 58. 59, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 72,73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88. 89, 90,91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1×10³, 2×10³,3×10³, 4×10³, 5×10³, 6×10³, 7×10³, 8×10³, 9×10³, 1×10⁴, 2×10⁴, 3×10⁴,4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵,5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶,6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷,7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸,8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹,9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰,9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹,9×10¹¹, and/or 1×10¹² Veillonella bacterium. In some embodiments, thepharmaceutical composition comprises no more than 1 Veillonella EVparticle for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8,8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5,9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, 20, 21, 22,23, 24, 25, 26, 27, 28. 29, 30, 31, 32, 33, 34, 35, 36, 37, 38. 39, 40,41, 42, 43, 44, 45, 46, 47, 48. 49, 50, 51, 52, 53, 54, 55, 56, 57, 58.59, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 72, 73, 74, 75, 76,77, 78. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88. 89, 90, 91, 92, 93, 94,95, 96, 97, 98. 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1×10³, 2×10³, 3×10³, 4×10³,5×10³, 6×10³, 7×10³, 8×10³, 9×10³, 1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴,6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵,7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶,8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷,9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸,1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰,2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹,2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, and/or1×10¹² Veillonella bacterium.

In certain embodiments, the bacterial composition suppresses the immuneresponse in delayed-type hypersensitivity (DTH). In certain embodiments,the bacterial composition induces a regulatory T cell or ananti-inflammatory response. In certain embodiments, the bacterialcomposition inhibits antigen-specific responses. In certain embodiments,the bacterial composition treats allergic contact dermatitis. In certainembodiments, the bacterial composition treats autoimmune myocarditis. Incertain embodiments, the bacterial composition treats diabetes mellitustype 1. In certain embodiments, the bacterial composition treatsgranulomas. In certain embodiments, the bacterial composition treatsperipheral neuropathies. In certain embodiments, the bacterialcomposition treats Hashimoto's thyroiditis. In certain embodiments, thebacterial composition treats multiple sclerosis. In certain embodiments,the bacterial composition treats rheumatoid arthritis.

In certain embodiments, the bacterial composition treats inflammation ofthe colon. In certain embodiments, the bacterial composition treatscolitis. Colitis may be acute and self-limited or long-term. In certainembodiments, the bacterial composition treats ulcerative colitis. Incertain embodiments, the bacterial composition treats digestivediseases. In certain embodiments, the bacterial composition treatsCrohn's disease. In certain embodiments, the bacterial compositiontreats inflammatory bowel disease (IBD). In certain embodiments, thebacterial composition treats microscopic colitis. In certainembodiments, the bacterial composition treats collagenous colitis. Incertain embodiments, the bacterial composition treats diversion colitis.In certain embodiments, the bacterial composition treats chemicalcolitis. In certain embodiments, the bacterial composition treatsischemic colitis. In certain embodiments, the bacterial compositiontreats indeterminate colitis. In certain embodiments, the bacterialcomposition treats atypical colitis. In some embodiments, the methodfurther comprises administering to the subject an additional therapeutic(e.g., an antibiotic, an immune suppressant, an anti-inflammatoryagent). In some embodiments, the method further comprises administeringto the subject is a second therapeutic bacterium.

In some embodiments, the subject is a mammal. In some embodiments, thesubject is a human. In some embodiments, the subject is a non-humanmammal (e.g., a dog, a cat, a cow, a horse, a pig, a donkey, a goat, acamel, a mouse, a rat, a guinea pig, a sheep, a llama, a monkey, agorilla or a chimpanzee).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the efficacy of orally administered Veillonella Strains A,B, and C in reducing antigen-specific ear swelling (ear thickness) at 24hours compared to vehicle (negative control), anti-inflammatoryDexamethasone (positive control), and Bifidobacterium animalis lactis ina KLH-based delayed type hypersensitivity mouse model.

FIG. 2 shows the efficacy of orally administered irradiated (25kGy)Veillonella Strain B in reducing antigen-specific ear swelling (earthickness) at 24 hours compared to vehicle (negative control) followingantigen challenge in a KLH-based delayed type hypersensitivity model.Both live and irradiated Veillonella Strain B were efficacious atinhibiting ear swelling, but irradiated Veillonella Strain B was evenmore efficacious than live Strain B, and it was even more efficaciousthan the positive control, Dexamethasone. Both viable and irradiatedVeillonella Strain C groups demonstrated efficacy but, unlike theirradiation of Strain B, the irradiation of Veillonella Strain C neitherenhanced nor diminished its efficacy.

FIG. 3 shows the efficacy of orally administered irradiated (25kGy)Veillonella Strain B in reducing antigen-specific ear swelling (earthickness) at 48 hours compared to vehicle (negative control) followingantigen challenge in a KLH-based delayed type hypersensitivity model.Both live and irradiated Veillonella Strain B were efficacious atinhibiting ear swelling, but irradiated Veillonella Strain B was evenmore efficacious than live Strain B, and it was even more efficaciousthan the positive control, Dexamethasone. Both viable and irradiatedVeillonella Strain C groups demonstrated efficacy but, unlike theirradiation of Strain B, the irradiation of Veillonella Strain C neitherenhanced nor diminished its efficacy.

FIG. 4 shows the efficacy of Veillonella Strains A and B EVs compared tointraperitoneal injected (i.p.) anti-PD-1 or vehicle in a mousecolorectal carcinoma model.

FIG. 5 shows the efficacy of Veillonella Strains A and B EVs compared tointraperitoneal injected (i.p.) anti-PD-1 or vehicle in a mousecolorectal carcinoma model at day 11.

FIG. 6 shows the dose and route of administration dependent efficacy ofVeillonella Strains A and B EVs compared to intraperitoneal injected(i.p.) anti-PD-1 or vehicle in a mouse colorectal carcinoma model.

FIG. 7 shows the dose and route of administration dependent efficacy ofVeillonella Strains A and B EVs compared to intraperitoneal injected(i.p.) anti-PD-1 or vehicle in a mouse colorectal carcinoma model at day11.

FIG. 8 shows the efficacy of administered irradiated (25kGy) VeillonellaStrains D, E, F and G in reducing antigen-specific ear swelling (earthickness) at 24 hours compared to vehicle (negative control) andanti-inflammatory Dexamethasone (positive control) in a KLH-baseddelayed type hypersensitivity mouse model.

FIG. 9 shows the efficacy of irradiated (25kGy) Veillonella Strains B,E, F, and G in reducing antigen-specific ear inflammation at 24 hourscompared to vehicle (negative control) and anti-inflammatoryDexamethasone (positive control) following antigen challenge in aKLH-based delayed type hypersensitivity model. Both live and irradiatedVeillonella Strain B and E were efficacious at inhibiting earinflammation, but irradiated Veillonella Strain E was even moreefficacious than live Strain E. For Veillonella Strain F, irradiationgamma-irradiation caused a non-performing strain of Veillonella tobecome efficacious. All the groups received 10 mg of the powder perdose.

FIG. 10 shows that Veillonella Strain C—was efficacious at reducing theNASH activity score (NAS) in mice receiving a methionine cholinedeficient (MCD) diet, which induces NASH symptoms.

FIG. 11 shows that Veillonella Strain C reduced Fibrosis in mice thatwere fed an MCD diet.

FIG. 12 shows that Veillonella Strain C reduced Hepatic TotalCholesterol in mice that were fed an MCD diet.

FIG. 13 shows that Veillonella Strain C reduced Hepatic Triglycerides inmice that were fed an MCD diet.

FIG. 14 shows the efficacy of Veillonella parvula Strain A EVs andVeillonella atypica Strains A and B EVs compared to that ofintraperitoneally (i.p.) administered anti-PD-1 or vehicle in a mousecolorectal carcinoma model at day 11. Welch's test is performed fortreatment vs. vehicle.

FIG. 15 shows the efficacy of Veillonella parvula Strain A EVs andVeillonella atypica Strains A and B EVs compared to that ofintraperitoneally (i.p.) administered anti-PD-1 or vehicle in a mousecolorectal carcinoma model at day 11. Welch's test is performed fortreatment vs. anti-PD-1.

DETAILED DESCRIPTION General

In certain aspects, provided herein are methods and compositions relatedto the treatment and/or prevention of disease (e.g., cancer, autoimmunedisease, inflammatory disease, metabolic disease), in a subject (e.g., ahuman subject) comprising administering a bacterial compositioncomprising Veillonella bacteria (e.g., Veillonella tobetsuensis,Veillonella parvula) as well as methods of making and/or identifyingsuch a bacterium.

Definitions

“Adjuvant” or “Adjuvant therapy” broadly refers to an agent that affectsan immunological or physiological response in a patient or subject. Forexample, an adjuvant might increase the presence of an antigen over timeor to an area of interest like a tumor, help absorb an antigenpresenting cell antigen, activate macrophages and lymphocytes andsupport the production of cytokines. By changing an immune response, anadjuvant might permit a smaller dose of an immune interacting agent toincrease the effectiveness or safety of a particular dose of the immuneinteracting agent. For example, an adjuvant might prevent T cellexhaustion

“Administration” broadly refers to a route of administration of acomposition to a subject. Examples of routes of administration includeoral administration, rectal administration, topical administration,inhalation (nasal) or injection. Administration by injection includesintravenous (IV), intramuscular (IM), intratumoral (IT) and subcutaneous(SC) administration. The pharmaceutical compositions described hereincan be administered in any form by any effective route, including butnot limited to intratumoral, oral, parenteral, enteral, intravenous,intraperitoneal, topical, transdermal (e.g., using any standard patch),intradermal, ophthalmic, (intra)nasally, local, non-oral, such asaerosol, inhalation, subcutaneous, intramuscular, buccal, sublingual,(trans)rectal, vaginal, intra-arterial, and intrathecal, transmucosal(e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal(e.g., trans- and perivaginally), intravesical, intrapulmonary,intraduodenal, intragastrical, and intrabronchial. In preferredembodiments, the pharmaceutical compositions described herein areadministered orally, rectally, intratumorally, topically,intravesically, by injection into or adjacent to a draining lymph node,intravenously, by inhalation or aerosol, or subcutaneously.

As used herein, the term “antibody” may refer to both an intact antibodyand an antigen binding fragment thereof. Intact antibodies areglycoproteins that include at least two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds. Each heavy chain includesa heavy chain variable region (abbreviated herein as V_(H)) and a heavychain constant region. Each light chain includes a light chain variableregion (abbreviated herein as V_(L)) and a light chain constant region.The V_(H) and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of theheavy and light chains contain a binding domain that interacts with anantigen. The term “antibody” includes, for example, monoclonalantibodies, polyclonal antibodies, chimeric antibodies, humanizedantibodies, human antibodies, multispecific antibodies (e.g., bispecificantibodies), single-chain antibodies and antigen-binding antibodyfragments.

The terms “antigen binding fragment” and “antigen-binding portion” of anantibody, as used herein, refers to one or more fragments of an antibodythat retain the ability to bind to an antigen. Examples of bindingfragments encompassed within the term “antigen-binding fragment” of anantibody include Fab, Fab′, F(ab′)₂, Fv, scFv, disulfide linked Fv, Fd,diabodies, single-chain antibodies, NANOBODIES®, isolated CDRH3, andother antibody fragments that retain at least a portion of the variableregion of an intact antibody. These antibody fragments can be obtainedusing conventional recombinant and/or enzymatic techniques and can bescreened for antigen binding in the same manner as intact antibodies.

“Cancer” broadly refers to an uncontrolled, abnormal growth of a host'sown cells leading to invasion of surrounding tissue and potentiallytissue distal to the initial site of abnormal cell growth in the host.Major classes include carcinomas which are cancers of the epithelialtissue (e.g., skin, squamous cells); sarcomas which are cancers of theconnective tissue (e.g., bone, cartilage, fat, muscle, blood vessels,etc.); leukemias which are cancers of blood forming tissue (e.g., bonemarrow tissue); lymphomas and myelomas which are cancers of immunecells; and central nervous system cancers which include cancers frombrain and spinal tissue. “Cancer(s),” “neoplasm(s),” and “tumor(s)” areused herein interchangeably. As used herein, “cancer” refers to alltypes of cancer or neoplasm or malignant tumors including leukemias,carcinomas and sarcomas, whether new or recurring. Specific examples ofcancers are: carcinomas, sarcomas, myelomas, leukemias, lymphomas andmixed type tumors. Non-limiting examples of cancers are new or recurringcancers of the brain, melanoma, bladder, breast, cervix, colon, head andneck, kidney, lung, non-small cell lung, mesothelioma, ovary, prostate,sarcoma, stomach, uterus and medulloblastoma.

The term “LPS mutant or lipopolysaccharide mutant” broadly refers toselected bacteria that comprises loss of LPS. Loss of LPS might be dueto mutations or disruption to genes involved in lipid A biosynthesis,such as lpxA, lpxC, and lpxD. Bacteria comprising LPS mutants can beresistant to aminoglycosides and polymyxins (polymyxin B and colistin).

“Cellular augmentation” broadly refers to the influx of cells orexpansion of cells in an environment that are not substantially presentin the environment prior to administration of a composition and notpresent in the composition itself. Cells that augment the environmentinclude immune cells, stromal cells, bacterial and fungal cells.Environments of particular interest are the microenvironments wherecancer cells reside or locate. In some instances, the microenvironmentis a tumor microenvironment or a tumor draining lymph node. In otherinstances, the microenvironment is a pre-cancerous tissue site or thesite of local administration of a composition or a site where thecomposition will accumulate after remote administration.

“Clade” refers to the OTUs or members of a phylogenetic tree that aredownstream of a statistically valid node in a phylogenetic tree. Theclade comprises a set of terminal leaves in the phylogenetic tree thatis a distinct monophyletic evolutionary unit and that share some extentof sequence similarity. “Operational taxonomic units,” “OTU” (or plural,“OTUs”) refer to a terminal leaf in a phylogenetic tree and is definedby a nucleic acid sequence, e.g., the entire genome, or a specificgenetic sequence, and all sequences that share sequence identity to thisnucleic acid sequence at the level of species. In some embodiments thespecific genetic sequence may be the 16S sequence or a portion of the16S sequence. In other embodiments, the entire genomes of two entitiesare sequenced and compared. In another embodiment, select regions suchas multilocus sequence tags (MLST), specific genes, or sets of genes maybe genetically compared. In 16S embodiments, OTUs that share ≥97%average nucleotide identity across the entire 16S or some variableregion of the 16S are considered the same OTU (see e.g. Claesson M J,Wang Q, O'Sullivan O, Greene-Diniz R, Cole J R, Ros R P. and O'Toole PW. 2010. Comparison of two next-generation sequencing technologies forresolving highly complex microbiota composition using tandem variable165 rRNA gene regions. Nucleic Acids Res 38: e200. Konstantinidis K T,Ramette A, and Tiedje J M. 2006. The bacterial species definition in thegenomic era. Philos Trans R Soc Lond B Biol Sci 361: 1929-1940.). Inembodiments involving the complete genome, MLSTs, specific genes, orsets of genes OTUs that share ≥95% average nucleotide identity areconsidered the same OTU (see e.g. Achtman M, and Wagner M. 2008.Microbial diversity and the genetic nature of microbial species. NatRev. Microbiol. 6: 431-440, Konstantinidis K T, Ramette A, and Tiedje JM, 2006. The bacterial species definition in the genomic era. PhilosTrans R Soc Lond B Biol Sci 361: 1929-1940.), OTUs are frequentlydefined by comparing sequences between organisms. Generally, sequenceswith less than 95% sequence identity are not considered to form part ofthe same OTU. OTUs may also be characterized by any combination ofnucleotide markers or genes, in particular highly conserved genes (e.g.,“house-keeping” genes), or a combination thereof. Such characterizationemploys, e.g., WGS data or a whole genome sequence.

A “combination” of two or more monoclonal microbial strains includes thephysical co-existence of the two monoclonal microbial strains, either inthe same material or product or in physically connected products, aswell as the temporal co-administration or co-localization of themonoclonal microbial strains.

The term “decrease” or “deplete” means a change, such that thedifference is, depending on circumstances, at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 1/100, 1/1000, 1/10,000, 1/100,000, 1/1,000,000or undetectable after treatment when compared to a pre-treatment state.

The term “ecological consortium” is a group of bacteria which tradesmetabolites and positively co-regulates one another, in contrast to twobacteria which induce host synergy through activating complementary hostpathways for improved efficacy.

The term “epitope” means a protein determinant capable of specificbinding to an antibody. Epitopes usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chains.Certain epitopes can be defined by a particular sequence of amino acidsto which an antibody is capable of binding.

As used herein, “engineered bacteria” are any bacteria that have beengenetically altered from their natural state by human intervention andthe progeny of any such bacteria. Engineered bacteria include, forexample, the products of targeted genetic modification, the products ofrandom mutagenesis screens and the products of directed evolution.

The term “gene” is used broadly to refer to any nucleic acid associatedwith a biological function. The term “gene” applies to a specificgenomic sequence, as well as to a cDNA or an mRNA encoded by thatgenomic sequence.

“Identity” as between nucleic acid sequences of two nucleic acidmolecules can be determined as a percentage of identity using knowncomputer algorithms such as the “FASTA” program, using for example, thedefault parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci.USA 85:2444 (other programs include the GCG program package (Devereux,J., et al., Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN,FASTA Atschul, S. F., et al., J Molec Biol 215:403 (1990); Guide to HugeComputers, Mrtin J. Bishop, ed., Academic Press, San Diego, 1994, andCarillo et al. (1988) SIAM J Applied Math 48:1073). For example, theBLAST function of the National Center for Biotechnology Informationdatabase can be used to determine identity. Other commercially orpublicly available programs include, DNAStar “MegAlign” program(Madison, Wis.) and the University of Wisconsin Genetics Computer Group(UWG) “Gap” program (Madison Wis.)).

As used herein, the term “immune disorder” refers to any disease,disorder or disease symptom caused by an activity of the immune system,including autoimmune diseases, inflammatory diseases and allergies.Immune disorders include, but are not limited to, autoimmune diseases(e.g., Lupus, Scleroderma, hemolytic anemia, vasculitis, type onediabetes, Grave's disease, rheumatoid arthritis, multiple sclerosis,Goodpasture's syndrome, pernicious anemia and/or myopathy), inflammatorydiseases (e.g., acne vulgaris, asthma, celiac disease, chronicprostatitis, glomerulonephritis, inflammatory bowel disease, pelvicinflammatory disease, reperfusion injury, rheumatoid arthritis,sarcoidosis, transplant rejection, vasculitis and/or interstitialcystitis), and/or an allergies (e.g., food allergies, drug allergiesand/or environmental allergies).

The term “increase” means a change, such that the difference is,depending on circumstances, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 2-fold, 4-fold, 10-fold, 100-fold, 10{circumflex over ( )}9fold, 10{circumflex over ( )}4 fold, 10{circumflex over ( )}5 fold,10{circumflex over ( )}6 fold, and/or 10{circumflex over ( )}7 foldgreater after treatment when compared to a pre-treatment state.Properties that may be increased include immune cells, bacterial cells,stromal cells, myeloid derived suppressor cells, fibroblasts,metabolites, and cytokines.

The “internal transcribed spacer” or “ITS” is a piece of non-functionalRNA located between structural ribosomal RNAs (rRNA) on a commonprecursor transcript often used for identification of eukaryotic speciesin particular fungi. The rRNA of fungi that forms the core of theribosome is transcribed as a signal gene and consists of the 8S, 5.8Sand 28S regions with ITS4 and 5 between the 8S and 5.8S and 5.8S and 28Sregions, respectively. These two intercistronic segments between the 18Sand 5.8S and 5.8S and 28S regions are removed by splicing and containsignificant variation between species for barcoding purposes aspreviously described (Schoch et al Nuclear ribosomal internaltranscribed spacer (ITS) region as a universal DNA barcode marker forFungi. PNAS 109:6241-6246. 2012). 18S rDNA is traditionally used forphylogenetic reconstruction however the ITS can serve this function asit is generally highly conserved but contains hypervariable regions thatharbor sufficient nucleotide diversity to differentiate genera andspecies of most fungus.

The term “isolated” or “enriched” encompasses a microbe, bacteria orother entity or substance that has been (1) separated from at least someof the components with which it was associated when initially produced(whether in nature or in an experimental setting), and/or (2) produced,prepared, purified, and/or manufactured by the hand of man. Isolatedmicrobes may be separated from at least about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, ormore of the other components with which they were initially associated.In some embodiments, isolated microbes are more than about 80%, about85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98%, about 99%, or more than about 99% pure.As used herein, a substance is “pure” if it is substantially free ofother components. The terms “purify,” “purifying” and “purified” referto a microbe or other material that has been separated from at leastsome of the components with which it was associated either wheninitially produced or generated (e.g., whether in nature or in anexperimental setting), or during any time after its initial production.A microbe or a microbial population may be considered purified if it isisolated at or after production, such as from a material or environmentcontaining the microbe or microbial population, and a purified microbeor microbial population may contain other materials up to about 10%,about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about80%, about 90%, or above about 90% and still be considered “isolated.”In some embodiments, purified microbes or microbial population are morethan about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, ormore than about 99% pure. In the instance of microbial compositionsprovided herein, the one or more microbial types present in thecomposition can be independently purified from one or more othermicrobes produced and/or present in the material or environmentcontaining the microbial type. Microbial compositions and the microbialcomponents thereof are generally purified from residual habitatproducts.

“Metabolite” as used herein refers to any and all molecular compounds,compositions, molecules, ions, co-factors, catalysts or nutrients usedas substrates in any cellular or microbial metabolic reaction orresulting as product compounds, compositions, molecules, ions,co-factors, catalysts or nutrients from any cellular or microbialmetabolic reaction.

“Microbe” refers to any natural or engineered organism characterized asa bacterium, fungus, microscopic alga, protozoan, and the stages ofdevelopment or life cycle stages (e.g., vegetative, spore (includingsporulation, dormancy, and germination), latent, biofilm) associatedwith the organism. Examples of gut microbes include: Actinomycesgraevenitzii, Actinomyces odontolyticus, Akkermansia muciniphila,Bacteroides caccae, Bacteroides fragilis, Bacteroides putredinis,Bacteroides thetaiotaomicron, Bacteroides vultagus, Bifidobacteriumadolescentis, Bifidobacterium bifidum, Bilophila wadsworthia,Lactococcus lactis, Butyrivibrio, Campylobacter gracilis, Clostridiacluster III, Clostridia cluster IV, Clostridia cluster IX(Acidaminococcaceae group), Clostridia cluster XI, Clostridia clusterXIII (Peptostreptococcus group), Clostridia cluster XIV, Clostridiacluster XV, Collinsella aerofaciens, Coprococcus, Corynebacteriumsunsvallense, Desulfomonas pigra, Dorea formicigenerans, Dorealongicatena, Escherichia coli, Eubacterium hadrum, Eubacterium rectale,Faecalibacteria prausnitzii, Gemella, Lactococcus, Lanchnospira,Mollicutes cluster XVI, Mollicutes cluster XVIII, Prevotella, Rothiamucilaginosa, Ruminococcus callidus, Ruminococcus gnavus, Ruminococcustorques, and Streptococcus.

“Microbiome” broadly refers to the microbes residing on or in body siteof a subject or patient. Microbes in a microbiome may include bacteria,viruses, eukaryotic microorganisms, and/or viruses. Individual microbesin a microbiome may be metabolically active, dormant, latent, or existas spores, may exist planktonically or in biofilms, or may be present inthe microbiome in sustainable or transient manner. The microbiome may bea commensal or healthy-state microbiome or a disease-state microbiome.The microbiome may be native to the subject or patient, or components ofthe microbiome may be modulated, introduced, or depleted due to changesin health state (e.g., precancerous or cancerous state) or treatmentconditions (e.g., antibiotic treatment, exposure to different microbes).In some aspects, the microbiome occurs at a mucosal surface. In someaspects, the microbiome is a gut microbiome. In some aspects, themicrobiome is a tumor microbiome.

A “microbiome profile” or a “microbiome signature” of a tissue or samplerefers to an at least partial characterization of the bacterial makeupof a microbiome. In some embodiments, a microbiome profile indicateswhether at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more bacterial strainsare present or absent in a microbiome.

“Modified” in reference to a bacteria broadly refers to a bacteria thathas undergone a change from its wild-type form. Examples of bacterialmodifications include genetic modification, gene expression, phenotypemodification, formulation, chemical modification, and dose orconcentration. Examples of improved properties are described throughoutthis specification and include, e.g., attenuation, auxotrophy, homing,or antigenicity. Phenotype modification might include, by way ofexample, bacteria growth in media that modify the phenotype of abacterium that increase or decrease virulence.

As used herein, a gene is “overexpressed” in a bacteria if it isexpressed at a higher level in an engineered bacteria under at leastsome conditions than it is expressed by a wild-type bacteria of the samespecies under the same conditions. Similarly, a gene is “underexpressed”in a bacteria if it is expressed at a lower level in an engineeredbacteria under at least some conditions than it is expressed by awild-type bacteria of the same species under the same conditions.

The terms “polynucleotide” and “nucleic acid” are used interchangeably.They refer to a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Polynucleotides may have any three-dimensional structure, and mayperform any function. The following are non-limiting examples ofpolynucleotides: coding or non-coding regions of a gene or genefragment, loci (locus) defined from linkage analysis, exons, introns,messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. A polynucleotide may comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs. Ifpresent, modifications to the nucleotide structure may be impartedbefore or after assembly of the polymer. A polynucleotide may be furthermodified, such as by conjugation with a labeling component. In allnucleic acid sequences provided herein, U nucleotides areinterchangeable with T nucleotides.

“Operational taxonomic units” and “OTU(s)” refer to a terminal leaf in aphylogenetic tree and is defined by a nucleic acid sequence, e.g., theentire genome, or a specific genetic sequence, and all sequences thatshare sequence identity to this nucleic acid sequence at the level ofspecies. In some embodiments the specific genetic sequence may be the16S sequence or a portion of the 16S sequence. In other embodiments, theentire genomes of two entities are sequenced and compared. In anotherembodiment, select regions such as multilocus sequence tags (MLST),specific genes, or sets of genes may be genetically compared. For 16S,OTUs that share ≥97% average nucleotide identity across the entire 16Sor some variable region of the 16S are considered the same OTU. See e.g.Claesson M J, Wang Q, O'Sullivan O, Greene-Diniz R, Cole J R, Ross R P,and O'Toole P W. 2010. Comparison of two next-generation sequencingtechnologies for resolving highly complex microbiota composition usingtandem variable 16S rRNA gene regions. Nucleic Acids Res 38: e200.Konstantinidis K T, Ramette A, and Tiedje J M. 2006. The bacterialspecies definition in the genomic era. Philos Trans R Soc Lond B BiolSci 361: 1929-1940. For complete genomes, MLSTs, specific genes, otherthan 16S, or sets of genes OTUs that share ≥95% average nucleotideidentity are considered the same OTU. See e.g., Achtman M, and Wagner M.2008. Microbial diversity and the genetic nature of microbial species.Nat. Rev. Microbiol. 6: 431-440. Konstantinidis K T, Ramette A, andTiedje J M. 2006. The bacterial species definition in the genomic era.Philos Trans R Soc Lond B Biol Sci 361: 1929-1940. OTUs are frequentlydefined by comparing sequences between organisms. Generally, sequenceswith less than 95% sequence identity are not considered to form part ofthe same OTU. OTUs may also be characterized by any combination ofnucleotide markers or genes, in particular highly conserved genes (e.g.,“house-keeping” genes), or a combination thereof. Operational TaxonomicUnits (OTUs) with taxonomic assignments made to, e.g., genus, species,and phylogenetic clade are provided herein.

As used herein, a substance is “pure” if it is substantially free ofother components. The terms “purify,” “purifying” and “purified” referto a EV or other material that has been separated from at least some ofthe components with which it was associated either when initiallyproduced or generated (e.g., whether in nature or in an experimentalsetting), or during any time after its initial production. An EV may beconsidered purified if it is isolated at or after production, such asfrom one or more other bacterial components, and a purified microbe ormicrobial population may contain other materials up to about 10%, about20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,about 90%, or above about 90% and still be considered “purified.” Insome embodiments, purified EVs are more than about 80%, about 85%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99%, or more than about 99% pure. EVcompositions and the microbial components thereof are, e.g., purifiedfrom residual habitat products.

As used herein, the term “purified EV composition” or “EV composition”refer to a preparation that includes EVs that have been separated fromat least one associated substance found in a source material (e.g.separated from at least one other bacterial component) or any materialassociated with the EVs in any process used to produce the preparation.It also refers to a composition that has been significantly enriched orconcentrated. In some embodiments the EVs are concentrated by 2 fold,3-fold, 4-fold, 5-fold, 10-fold, 100-fold, 1000-fold, 10,000-fold ormore than 10,000 fold.

As used herein, “specific binding” refers to the ability of an antibodyto bind to a predetermined antigen or the ability of a polypeptide tobind to its predetermined binding partner. Typically, an antibody orpolypeptide specifically binds to its predetermined antigen or bindingpartner with an affinity corresponding to a K_(D) of about 10⁻⁷ M orless, and binds to the predetermined antigen/binding partner with anaffinity (as expressed by K_(D)) that is at least 10 fold less, at least100 fold less or at least 1000 fold less than its affinity for bindingto a non-specific and unrelated antigen/binding partner (e.g., BSA,casein). Alternatively, specific binding applies more broadly to a twocomponent system where one component is a protein, lipid, orcarbohydrate or combination thereof and engages with the secondcomponent which is a protein, lipid, carbohydrate or combination thereofin a specific way.

The terms “subject” or “patient” refers to any animal. A subject or apatient described as “in need thereof” refers to one in need of atreatment for a disease. Mammals (i.e., mammalian animals) includehumans, laboratory animals (e.g., primates, rats, mice), livestock(e.g., cows, sheep, goats, pigs), and household pets (e.g., dogs, cats,rodents). For example, the subject may be a non-human mammal includingbut not limited to of a dog, a cat, a cow, a horse, a pig, a donkey, agoat, a camel, a mouse, a rat, a guinea pig, a sheep, a llama, a monkey,a gorilla or a chimpanzee. The subject or patient may be healthy, or maybe suffering from an immune disorder at any developmental stage.

“Strain” refers to a member of a bacterial species with a geneticsignature such that it may be differentiated from closely-relatedmembers of the same bacterial species. The genetic signature may be theabsence of all or part of at least one gene, the absence of all or partof at least on regulatory region (e.g., a promoter, a terminator, ariboswitch, a ribosome binding site), the absence (“curing”) of at leastone native plasmid, the presence of at least one recombinant gene, thepresence of at least one mutated gene, the presence of at least oneforeign gene (a gene derived from another species), the presence atleast one mutated regulatory region (e.g., a promoter, a terminator, ariboswitch, a ribosome binding site), the presence of at least onenon-native plasmid, the presence of at least one antibiotic resistancecassette, or a combination thereof. Genetic signatures between differentstrains may be identified by PCR amplification optionally followed byDNA sequencing of the genomic region(s) of interest or of the wholegenome. In the case in which one strain (compared with another of thesame species) has gained or lost antibiotic resistance or gained or losta biosynthetic capability (such as an auxotrophic strain), strains maybe differentiated by selection or counter-selection using an antibioticor nutrient/metabolite, respectively.

As used herein, the term “treating” a disease in a subject or “treating”a subject having or suspected of having a disease refers to subjectingthe subject to a pharmaceutical treatment, e.g., the administration ofone or more agents, such that at least one symptom of the disease isdecreased or prevented from worsening. Thus, in one embodiment,“treating” refers inter alia to delaying progression, expeditingremission, inducing remission, augmenting remission, speeding recovery,increasing efficacy of or decreasing resistance to alternativetherapeutics, or a combination thereof.

Bacteria

In certain aspects, provided herein are methods of using a bacterialcomposition comprising Veillonella bacteria and/or a product of suchbacteria (e.g., extracellular vesicles (EVs) and/or pharmaceuticallyactive biomasses (PhABs)). In some embodiments, the Veillonella bacteriais of the following species: Veillonella tobetsuensis or Veillonellaparvula. In some embodiments, the Veillonella bacteria is of thefollowing species: Veillonella atypica or Veillonella dispar. In someembodiments, the bacteria is a strain of bacteria listed in Table 1.

TABLE 1 Bacterial Strains SEQ ID Strain Organism name 16S sequence NO AVeillonella >S11-19-357F SEQ ID tobetsuensisAGCAACGCCGCGTGAGTGATGACGGCCTTCGGGTT NO 1GTAAAGCTCTGTTAATCGGGACGAAAGGCCTTCTTG CGAATAGTTAGAAGGATTGACGGTACCGGAATAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTA ATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGATCGGTCAGTCTGT CTTAAAAGTTCGGGGCTTAACCCCGTGAGGGGATGGAAACTGCTGATCTAGAGTATCGGAGAGGAAAGTG GAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAAGAACACCAGTGGCGAAGGCGACTTTCTGGAC GAAAACTGACGCTGAGGCGCGAAAGCCAGGGGAGCGAACGGGATTAGATACCCCGGTAGTCCTGGCCGT AAACGATGGGTACTAGGTGTAGGAGGTATCGACCCCTTCTGTGCCGGAGTTAACGCAATAAGTACCCCGCC TGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTG GTTTAATTCGACGCAACGCGAAGAACCTTACCAGGTCTTGACATTGATGGACAGAACTAGAGATAGTTCCT CTTCTTCGGAAGCCAGAAAACAGGTGGTGCACGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCTTATGTTGCCAG CACTTCGGGTGGGAACTCAT BVeillonella >S14-201 Contig SEQ ID parvulaGAGTGATGACGGCCTTCGGGTTGTAAAGCTCTGTTA NO 2ATCGGGACGAAAGGCCTTCTTGCGAATAGTGAGAA GGATTGACGGTACCGGAATAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGC AAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGATAGGTCAGTCTGTCTTAAAAGTTCGG GGCTTAACCCCGTGATGGGATGGAAACTGCCAATCTAGAGTATCGGAGAGGAAAGTGGAATTCCTAGTGT AGCGGTGAAATGCGTAGATATTAGGAAGAACACCAGTGGCGAAGGCGACTTTCTGGACGAAAACTGACGC TGAGGCGCGAAAGCCAGGGGAGCGAACGGGATTAGATACCCCGGTAGTCCTGGCCGTAAACGATGGGTA CTAGGTGTAGGAGGTATCGACCCCTTCTGTGCCGGAGTTAACGCAATAAGTACCCCGCCTGGGGAGTACGA CCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGAC GCAACGCGAAGAACCTTACCAGGTCTTGACATTGATGGACAGAACCAGAGATGGTTCCTCTTCTTCGGAAG CCAGAAAACAGGTGGTGCACGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAG CGCAACCCCTATCTTATGTTGCCAGCACTTTGGGTGGGGACTCATGAGAGACTGCCGCAGACAATGCGGAG GAAGGCGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTACTACAATGGGAGTT AATAGACGGAAGCGAGATCGCGAGATGGAGCAAACCCGAGAAACACTCTCTCAGTTCGGATCGTAGGCTGCAACTCGCCTACGTGAAGTCGGAATCGCTAGTAATC GCAGGTCAGCATACTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAAAGTCG GAAGTGCCCAAAGCCGGTGGGGTAACCTTC CVeillonella >S14-205 Contig SEQ ID parvulaGAGTGATGACGGCCTTCGGGTTGTAAAGCTCTGTTA NO. 3ATCGGGACGAAAGGCCTTCTTGCGAATAGTGAGAA GGATTGACGGTACCGGAATAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGC AAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGATAGGTCAGTCTGTCTTAAAAGTTCGG GGCTTAACCCCGTGATGGGATGGAAACTGCCAATCTAGAGTATCGGAGAGGAAAGTGGAATTCCTAGTGT AGCGGTGAAATGCGTAGATATTAGGAAGAACACCAGTGGCGAAGGCGACTTTCTGGACGAAAACTGACGC TGAGGCGCGAAAGCCAGGGGAGCGAACGGGATTAGATACCCCGGTAGTCCTGGCCGTAAACGATGGGTA CTAGGTGTAGGAGGTATCGACCCCTTCTGTGCCGGAGTTAACGCAATAAGTACCCCGCCTGGGGAGTACGA CCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGAC GCAACGCGAAGAACCTTACCAGGTCTTGACATTGATGGACAGAACCAGAGATGGTTCCTCTTCTTCGGAAG CCAGAAAACAGGTGGTGCACGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAG CGCAACCCCTATCTTATGTTGCCAGCACTTTGGGTGGGGACTCATGAGAGACTGCCGCAGACAATGCGGAG GAAGGCGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTACTACAATGGGAGTT AATAGACGGAAGCGAGATCGCGAGATGGAGCAAACCCGAGAAACACTCTCTCAGTTCGGATCGTAGGCTGCAACTCGCCTACGTGAAGTCGGAATCGCTAGTAATC GCAGGTCAGCATACTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAAAGTCG GAAGTGCCCAAAGCCGGTG

In some embodiments, the bacteria is a strain comprising at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% sequence identity(e.g., at least 99.5% sequence identity, at least 99.6% sequenceidentity, at least 99.7% sequence identity, at least 99.8% sequenceidentity, at least 99.9% sequence identity) to the nucleotide sequence(e.g., genomic, 16S or CRISPR nucleotide sequence) of the bacterialstrains listed in Table 1.

Applicant represents that the ATCC is a depository affording permanenceof the deposit and ready accessibility thereto by the public if a patentis granted. All restrictions on the availability to the public of thematerial so deposited will be irrevocably removed upon the granting of apatent. The material will be available during the pendency of the patentapplication to one determined by the Commissioner to be entitled theretounder 37 CFR 1.14 and 35 U.S.C. 122. The deposited material will bemaintained with all the care necessary to keep it viable anduncontaminated for a period of at least five years after the most recentrequest for the furnishing of a sample of the deposited plasmid, and inany case, for a period of at least thirty (30) years after the date ofdeposit or for the enforceable life of the patent, whichever period islonger. Applicant acknowledges its duty to replace the deposit shouldthe depository be unable to furnish a sample when requested due to thecondition of the deposit.

In some embodiments, the bacteria described herein are modified toimprove colonization and/or engraftment in the mammaliangastrointestinal tract (e.g., modified metabolism, such as improvedmucin degradation, enhanced competition profile, increased motility,increased adhesion to gut epithelial cells, modified chemotaxis). Insome embodiments, the bacteria described herein are modified to enhancetheir immunomodulatory and/or therapeutic effect (e.g., either alone orin combination with another therapeutic agent). In some embodiments, thebacteria described herein are modified to enhance immune activation(e.g., through modified production of polysaccharides, pili, fimbriae,adhesins, vesicles). In some embodiments, the bacteria described hereinare modified to improve bacterial manufacturing (e.g., higher oxygentolerance, improved freeze-thaw tolerance, shorter generation times).

The Veillonella bacteria (e.g., Veillonella tobetsuensis, Veillonellaparvula) can be cultured according to methods known in the art. Forexample, the Veillonella bacteria (e.g., a strain of bacteria listed inTable 1) can be grown in ATCC Medium 2722, ATCC Medium 1490, or othermedium using methods disclosed, for example in Caballero et al., 2017.“Cooperating Commensals Restore Colonization Resistance toVancomycin-Resistant Enterococcus faecium” Cell Host & Microbe21:592-602, which is hereby incorporated by reference in its entirety.

Production of EVs

In certain aspects, the Veillonella bacteria (e.g., Veillonellatobetsuensis, Veillonella parvula) EVs described herein can be preparedusing any method known in the art.

In some embodiments, the immune modulating Veillonella bacteria (e.g.,Veillonella tobetsuensis, Veillonella parvula) EVs are prepared withoutan EV purification step. For example, in some embodiments, immunemodulating Veillonella bacteria (e.g., Veillonella tobetsuensis,Veillonella parvula) comprising the EVs described herein are killedusing a method that leaves the immune modulating Veillonella bacteria(e.g., Veillonella tobetsuensis, Veillonella parvula) EVs intact and theresulting bacterial components, including the EVs, are used in themethods and compositions described herein. In some embodiments, theimmune modulating Veillonella bacteria (e.g., Veillonella tobetsuensis,Veillonella parvula) are killed using an antibiotic (e.g., using anantibiotic described herein). In some embodiments, the immune modulatingVeillonella bacteria (e.g., Veillonella tobetsuensis, Veillonellaparvula) are killed using UV irradiation.

In some embodiments, the EVs described herein are purified from one ormore other bacterial components. Methods for purifying EVs from bacteriaare known in the art. In some embodiments EVs are prepared frombacterial cultures using methods described in S. Bin Park, et al. PLoSONE. 6(3):e17629 (2011) or G. Norheim, et al. PLoS ONE. 10(9): e0134353(2015), each of which is hereby incorporated by reference in itsentirety. In some embodiments, the bacteria are cultured to high opticaldensity and then centrifuged to pellet bacteria (e.g., at 10,000×g for30 min at 4° C., at 15,500×g for 15 min at 4° C.). In some embodiments,the culture supernatants are then passed through filters to excludeintact bacterial cells (e.g., a 0.22 μm filter). In some embodiments,the supernatants are then subjected to tangential flow filtration,during which the supernatant is concentrated, species smaller than 100kDa are removed, and the media is partially exchanged with PBS. In someembodiments, filtered supernatants are centrifuged to pellet bacterialEVs (e.g., at 100,000-150,000×g for 1-3 hours at 4° C., at 200,000×g for1-3 hours at 4° C.). In some embodiments, the EVs are further purifiedby resuspending the resulting EV pellets (e.g., in PBS), and applyingthe resuspended EVs to an Optiprep (iodixanol) gradient or gradient(e.g., a 30-60% discontinuous gradient, a 0-45% discontinuous gradient),followed by centrifugation (e.g., at 200,000×g for 4-20 hours at 4° C.).EV bands can be collected, diluted with PBS, and centrifuged to pelletthe EVs (e.g., at 150,000×g for 3 hours at 4° C., at 200,000×g for 1hour at 4° C.). The purified EVs can be stored, for example, at −80° C.or −20° C. until use. In some embodiments, the EVs are further purifiedby treatment with DNase and/or proteinase K.

For example, in some embodiments, cultures of immune modulatingVeillonella bacteria (e.g., Veillonella tobetsuensis, Veillonellaparvula) disclosed herein can be centrifuged at 11,000×g for 20-40 minat 4° C. to pellet bacteria. Culture supernatants may be passed througha 0.22 μm filter to exclude intact bacterial cells. Filteredsupernatants may then be concentrated using methods that may include,but are not limited to, ammonium sulfate precipitation,ultracentrifugation, or filtration. For example, for ammonium sulfateprecipitation, 1.5-3 M ammonium sulfate can be added to filteredsupernatant slowly, while stirring at 4° C. Precipitations can beincubated at 4° C. for 8-48 hours and then centrifuged at 11,000×g for20-40 min at 4° C. The resulting pellets contain immune modulatingVeillonella bacteria (e.g., Veillonella tobetsuensis, Veillonellaparvula) EVs and other debris. Using ultracentrifugation, filteredsupernatants can be centrifuged at 100,000-200,000×g for 1-16 hours at4° C. The pellet of this centrifugation contains immune modulatingVeillonella bacteria (e.g., Veillonella tobetsuensis, Veillonellaparvula) EVs and other debris such as large protein complexes. In someembodiments, using a filtration technique, such as through the use of anAmicon Ultra spin filter or by tangential flow filtration, supernatantscan be filtered so as to retain species of molecular weight >50 or 100kDa.

Alternatively, EVs can be obtained from immune modulating Veillonellabacteria (e.g., Veillonella tobetsuensis, Veillonella parvula) culturescontinuously during growth, or at selected time points during growth,for example, by connecting a bioreactor to an alternating tangentialflow (ATF) system (e.g., XCell ATF from Repligen). The ATF systemretains intact cells (>0.22 um) in the bioreactor, and allows smallercomponents (e.g., EVs, free proteins) to pass through a filter forcollection. For example, the system may be configured so that the <0.22um filtrate is then passed through a second filter of 100 kDa, allowingspecies such as EVs between 0.22 um and 100 kDa to be collected, andspecies smaller than 100 kDa to be pumped back into the bioreactor.Alternatively, the system may be configured to allow for medium in thebioreactor to be replenished and/or modified during growth of theculture. EVs collected by this method may be further purified and/orconcentrated by ultracentrifugation or filtration as described above forfiltered supernatants.

EVs obtained by methods provided herein may be further purified bysize-based column chromatography, by affinity chromatography, byion-exchange chromatography, and by gradient ultracentrifugation, usingmethods that may include, but are not limited to, use of a sucrosegradient or Optiprep gradient. Briefly, using a sucrose gradient method,if ammonium sulfate precipitation or ultracentrifugation were used toconcentrate the filtered supernatants, pellets are resuspended in 60%sucrose, 30 mM Tris, pH 8.0. If filtration was used to concentrate thefiltered supernatant, the concentrate is buffer exchanged into 60%sucrose, 30 mM Tris, pH 8.0, using an Amicon Ultra column. Samples areapplied to a 35-60% discontinuous sucrose gradient and centrifuged at200,000×g for 3-24 hours at 4° C. Briefly, using an Optiprep gradientmethod, if ammonium sulfate precipitation or ultracentrifugation wereused to concentrate the filtered supernatants, pellets are resuspendedin PBS and 3 volumes of 60% Optiprep are added to the sample. In someembodiments, if filtration was used to concentrate the filteredsupernatant, the concentrate is diluted using 60% Optiprep to a finalconcentration of 35% Optiprep. Samples are applied to a 0-45%discontinuous Optiprep gradient and centrifuged at 200,000×g for 3-24hours at 4° C., e.g. 4-24 hours at 4° C.

In some embodiments, to confirm sterility and isolation of the EVpreparations, EVs are serially diluted onto agar medium used for routineculture of the bacteria being tested, and incubated using routineconditions. Non-sterile preparations are passed through a 0.22 um filterto exclude intact cells. To further increase purity, isolated EVs may beDNase or proteinase K treated.

In some embodiments, for preparation of EVs used for in vivo injections,purified EVs are processed as described previously (G. Norheim, et al.PLoS ONE. 10(9): e0134353 (2015)). Briefly, after sucrose gradientcentrifugation, bands containing EVs are resuspended to a finalconcentration of 50 μg/mL in a solution containing 3% sucrose or othersolution suitable for in vivo injection known to one skilled in the art.This solution may also contain adjuvant, for example aluminum hydroxideat a concentration of 0-0.5% (w/v). In some embodiments, for preparationof EVs used for in vivo injections, EVs in PBS are sterile-filtered to<0.22 um.

In certain embodiments, to make samples compatible with further testing(e.g. to remove sucrose prior to TEM imaging or in vitro assays),samples are buffer exchanged into PBS or 30 mM Tris, pH 8.0 usingfiltration (e.g. Amicon Ultra columns), dialysis, or ultracentrifugation(200,000×g, ≥3 hours, 4° C.) and resuspension.

In some embodiments, the sterility of the EV preparations can beconfirmed by plating a portion of the EVs onto agar medium used forstandard culture of the bacteria used in the generation of the EVs andincubating using standard conditions.

In some embodiments select EVs are isolated and enriched bychromatography and binding surface moieties on EVs. In otherembodiments, select EVs are isolated and/or enriched by fluorescent cellsorting by methods using affinity reagents, chemical dyes, recombinantproteins or other methods known to one skilled in the art.

Bacterial/Pharmaceutical Compositions

In certain aspects, provided herein are bacterial compositionscomprising a Veillonella bacteria (e.g., Veillonella tobetsuensis,Veillonella parvula) and/or a product of such bacteria (e.g.,extracellular vesicles (EVs) and/or pharmaceutically active biomasses(PhABs)). In some embodiments, the bacteria is a strain of bacterialisted in Table 1. In some embodiments, the bacteria is a straincomprising at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% sequence identity (e.g., at least 99.5% sequence identity, atleast 99.6% sequence identity, at least 99.7% sequence identity, atleast 99.8% sequence identity, at least 99.9% sequence identity) to thenucleotide sequence of a bacterial strain listed in Table 1. In someembodiments, the bacterial formulation comprises a bacterium and/or acombination of bacteria described herein and a pharmaceuticallyacceptable carrier (e.g., a pharmaceutical composition).

In certain embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the bacteria inthe bacterial composition are Veillonella bacteria (e.g., a strain ofbacteria listed in Table 1). In certain embodiments, substantially allof the bacteria in the bacterial composition are Veillonella bacteria(e.g., a strain of bacteria listed in Table 1). In certain embodiments,the bacterial composition comprises at least 1×10³ colony forming units(CFUs), 1×10⁴ colony forming units (CFUs), 1×10⁵ colony forming units(CFUs), 5×10⁵ colony forming units (CFUs), 1×10⁶ colony forming units(CFUs), 2×10⁶ colony forming units (CFUs), 3×10⁶ colony forming units(CFUs), 4×10⁶ colony forming units (CFUs), 5×10⁶ colony forming units(CFUs), 6×10⁶ colony forming units (CFUs), 7×10⁶ colony forming units(CFUs), 8×10⁶ colony forming units (CFUs), 9×10⁶ colony forming units(CFUs), 1×10⁷ colony forming units (CFUs), 2×10⁷ colony forming units(CFUs), 3×10⁷ colony forming units (CFUs), 4×10⁷ colony forming units(CFUs), 5×10⁷ colony forming units (CFUs), 6×10⁷ colony forming units(CFUs), 7×10⁷ colony forming units (CFUs), 8×10⁷ colony forming units(CFUs), 9×10⁷ colony forming units (CFUs), 1×10⁸ colony forming units(CFUs), 2×10⁸ colony forming units (CFUs), 3×10⁸ colony forming units(CFUs), 4×10⁸ colony forming units (CFUs), 5×10⁸ colony forming units(CFUs), 6×10⁸ colony forming units (CFUs), 7×10⁸ colony forming units(CFUs), 8×10⁸ colony forming units (CFUs), 9×10⁸ colony forming units(CFUs), 1×10⁹ colony forming units (CFUs), 5×10⁹ colony forming units(CFUs), 1×10¹⁰ colony forming units (CFUs) 5×10¹⁰ colony forming units(CFUs), 1×10¹¹ colony forming units (CFUs) 5×10¹¹ colony forming units(CFUs), 1×10¹² colony forming units (CFUs) 5×10¹² colony forming units(CFUs), 1×10¹³ colony forming units (CFUs) of Veillonella bacteria(e.g., a strain of bacteria listed in Table 1).

In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%of the bacteria in the composition are selected from among the bacterialspecies described herein. 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of thebacteria in the composition are selected from among the bacterialstrains described herein.

In some embodiments, the compositions described herein may include onlyone species of bacteria described herein or may include two or morespecies of the bacteria described herein. For example, 1, 2, or 3 of thespecies described herein, in any combination, can be included in thecompositions provided herein.

In some embodiments, the bacterial composition comprises a killedbacterium, a live bacterium and/or an attenuated bacterium. Bacteria maybe heat-killed by pasteurization, sterilization, high temperaturetreatment, spray cooking and/or spray drying (heat treatments can beperformed at 50° C., 65° C., 85° C. or a variety of other temperaturesand/or a varied amount of time). Bacteria may also be killed orinactivated using γ-irradiation (gamma irradiation), exposure to UVlight, formalin-inactivation, and/or freezing methods, or a combinationthereof. For example, the bacteria may be exposed to 1, 2, 3, 4, 5, 10,15, 20, 25, 30, 35, 40, or 50kGy of radiation prior to administration.In some embodiments, bacteria (e.g., Veillonella tobetsuensis,Veillonella parvula) are killed using gamma irradiation. In someembodiments, the bacteria are killed or inactivated using electronirradiation (e.g., beta radiation) or x-ray irradiation.

Bacteria may be grown to various growth phases and tested for efficacyat different dilutions and at different points during the growth phase.For example, bacteria may be tested for efficacy followingadministration at stationary phase (including early or late stationaryphase), or at various timepoints during exponential phase in addition toinactivation by various methods, bacteria may be tested for efficacyusing different ratios of live versus inactivated cells, or differentratios of cells at various growth phases.

In certain embodiments, provided herein are pharmaceutical compositionscomprising Veillonella EVs (e.g., Veillonella tobetsuensis EVs,Veillonella parvula EVs) and/or Veillonella bacteria (e.g., Veillonellatobetsuensis, Veillonella parvula). provided herein (e.g., an EVcomposition), such as those disclosed in U.S. Provisional PatentApplication No. 62/578,559, hereby incorporated by reference in itsentirety. In some embodiments, the EV composition comprises an EV and/ora combination of EVs described herein and a pharmaceutically acceptablecarrier.

In some embodiments, the pharmaceutical compositions compriseVeillonella EVs substantially or entirely free of bacteria. In someembodiments, the pharmaceutical compositions comprise both VeillonellaEVs and whole Veillonella bacteria (e.g., live bacteria, killedbacteria, attenuated bacteria). In certain embodiments, thepharmaceutical compositions comprise Veillonella bacteria that issubstantially or entirely free of EVs.

In some embodiments, the pharmaceutical composition comprises at least 1Veillonella bacterium for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2,6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18.19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29, 30, 31, 32, 33, 34, 35, 36,37, 38. 39, 40, 41, 42, 43, 44, 45, 46, 47, 48. 49, 50, 51, 52, 53, 54,55, 56, 57, 58. 59, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 72,73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88. 89, 90,91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1×10³, 2×10³,3×10³, 4×10³, 5×10³, 6×10³, 7×10³, 8×10³, 9×10³, 1×10⁴, 2×10⁴, 3×10⁴,4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵,5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶,6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷,7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸,8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹,9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰,9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹,9×10¹¹, and/or 1×10¹² Veillonella EV particles.

In some embodiments, the pharmaceutical composition comprises about 1Veillonella bacterium for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2,6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18.19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29, 30, 31, 32, 33, 34, 35, 36,37, 38. 39, 40, 41, 42, 43, 44, 45, 46, 47, 48. 49, 50, 51, 52, 53, 54,55, 56, 57, 58. 59, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 72,73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88. 89, 90,91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1×10³, 2×10³,3×10³, 4×10³, 5×10³, 6×10³, 7×10³, 8×10³, 9×10³, 1×10⁴, 2×10⁴, 3×10⁴,4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵,5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶,6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷,7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸,8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹,9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰,9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹,9×10¹¹, and/or 1×10¹² Veillonella EV particles.

In certain embodiments, the pharmaceutical composition comprises acertain ratio of Veillonella bacteria particles to Veillonella EVparticles. The number of Veillonella bacteria particles can be based onactual particle number or (if the bacteria is live) the number of CFUs.The particle number can be established by combining a set number ofpurified Veillonella EVs with a set number of purified Veillonellabacterium, by modifying the growth conditions under which theVeillonella bacteria are cultured, or by modifying the Veillonellabacteria itself to produce more or fewer Veillonella EVs.

In some embodiments, to quantify the numbers of Veillonella EVs and/orVeillonella bacteria present in a bacterial sample, electron microscopy(e.g., EM of ultrathin frozen sections) can be used to visualize thevesicles and bacteria and count their relative numbers. Alternatively,combinations of nanoparticle tracking analysis (NTA), Coulter counting,and dynamic light scattering (DLS) or a combination of these techniquescan be used. NTA and the Coulter counter count particles and show theirsizes. DLS gives the size distribution of particles, but not theconcentration. Bacteria frequently have diameters of 1-2 um. The fullrange is 0.2-20 um. Combined results from Coulter counting and NTA canreveal the numbers of bacteria in a given sample. Coulter countingreveals the numbers of particles with diameters of 0.7-10 um. NTAreveals the numbers of particles with diameters of 50-1400 nm. For mostbacterial samples, the Coulter counter alone can reveal the number ofbacteria in a sample. EVs are 20-250 nm in diameter. NTA will allow usto count the numbers of particles that are 50-250 nm in diameter. DLSreveals the distribution of particles of different diameters within anapproximate range of 1 nm-3 um.

In some embodiments, the pharmaceutical composition comprises no morethan 1 Veillonella bacterium for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1,3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6,4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1,9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10, 11, 12, 13, 14, 15, 16, 17,18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29, 30, 31, 32, 33, 34, 35,36, 37, 38. 39, 40, 41, 42, 43, 44, 45, 46, 47, 48. 49, 50, 51, 52, 53,54, 55, 56, 57, 58. 59, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71,72, 73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88. 89,90, 91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1×10³,2×10³, 3×10³, 4×10³, 5×10³, 6×10³, 7×10³, 8×10³, 9×10³, 1×10⁴, 2×10⁴,3×10⁴, 4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵, 3×10⁵,4×10⁵, 5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶,5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷,6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸,7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹,8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰,8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹,8×10¹¹, 9×10¹¹, and/or 1×10¹² Veillonella EV particles.

In some embodiments, the pharmaceutical composition comprises at least 1Veillonella EV particle for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2,6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18.19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29, 30, 31, 32, 33, 34, 35, 36,37, 38. 39, 40, 41, 42, 43, 44, 45, 46, 47, 48. 49, 50, 51, 52, 53, 54,55, 56, 57, 58. 59, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 72,73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88. 89, 90,91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1×10³, 2×10³,3×10³, 4×10³, 5×10³, 6×10³, 7×10³, 8×10³, 9×10³, 1×10⁴, 2×10⁴, 3×10⁴,4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵,5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶,6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷,7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸,8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹,9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰,9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹,9×10¹¹, and/or 1×10¹² Veillonella bacterium.

In some embodiments, the pharmaceutical composition comprises about 1Veillonella EV particle for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2,6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18.19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29, 30, 31, 32, 33, 34, 35, 36,37, 38. 39, 40, 41, 42, 43, 44, 45, 46, 47, 48. 49, 50, 51, 52, 53, 54,55, 56, 57, 58. 59, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 72,73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88. 89, 90,91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1×10³, 2×10³,3×10³, 4×10³, 5×10³, 6×10³, 7×10³, 8×10³, 9×10³, 1×10⁴, 2×10⁴, 3×10⁴,4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵,5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶,6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷,7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸,8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹,9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰,9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹,9×10¹¹, and/or 1×10¹² Veillonella bacterium. In some embodiments, thepharmaceutical composition comprises no more than 1 Veillonella EVparticle for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8,8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5,9.6, 9.7, 9.8. 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, 20, 21, 22,23, 24, 25, 26, 27, 28. 29, 30, 31, 32, 33, 34, 35, 36, 37, 38. 39, 40,41, 42, 43, 44, 45, 46, 47, 48. 49, 50, 51, 52, 53, 54, 55, 56, 57, 58.59, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 72, 73, 74, 75, 76,77, 78. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88. 89, 90, 91, 92, 93, 94,95, 96, 97, 98. 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1×10³, 2×10³, 3×10³, 4×10³,5×10³, 6×10³, 7×10³, 8×10³, 9×10³, 1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴,6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵,7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶,8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷,9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸,1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰,2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹,2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, and/or1×10¹² Veillonella bacterium.

In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% of the particles in the pharmaceuticalcomposition are Veillonella EVs.

In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% of the particles in the pharmaceuticalcomposition are Veillonella bacteria.

In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% of the particles in the pharmaceuticalcomposition are Veillonella EVs.

In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% of the particles in the pharmaceuticalcomposition are Veillonella bacteria.

In some embodiments, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% of the particles in the pharmaceutical compositionare Veillonella EVs.

In some embodiments, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% of the particles in the pharmaceutical compositionare Veillonella bacteria.

In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% of the protein in the pharmaceuticalcomposition is Veillonella EV protein.

In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% of the protein in the pharmaceuticalcomposition is Veillonella bacteria protein.

In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% of the protein in the pharmaceuticalcomposition is Veillonella EV protein.

In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% of the protein in the pharmaceuticalcomposition is Veillonella bacteria protein.

In some embodiments, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% of the protein in the pharmaceutical composition isVeillonella EV protein.

In some embodiments, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% of the protein in the pharmaceutical composition isVeillonella bacteria protein.

In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% of the lipids in the pharmaceuticalcomposition are Veillonella EV lipids.

In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% of the lipids in the pharmaceuticalcomposition are Veillonella bacteria lipids.

In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% of the lipids in the pharmaceuticalcomposition are Veillonella EV lipids.

In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% of the lipids in the pharmaceuticalcomposition are Veillonella bacteria lipids.

In some embodiments, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% of the lipids in the pharmaceutical composition areVeillonella EV lipids.

In some embodiments, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% of the lipids in the pharmaceutical composition areVeillonella bacteria lipids.

In some embodiments, the Veillonella EVs in the pharmaceuticalcomposition are purified from one or more other bacterial components. Insome embodiments, the pharmaceutical composition further comprises otherbacterial components. In some embodiments, the pharmaceuticalcomposition comprise bacteria cells.

As described in detail below, the pharmaceutical compositions disclosedherein may be specially formulated for administration in solid or liquidform, including those adapted for oral or rectal administration.

In some embodiments, the composition described herein may be apharmaceutical composition, a dietary supplement, or a food product(e.g., a food or beverage). In some embodiments, the food product is ananimal feed.

In certain embodiments, the pharmaceutical composition for oraladministration described herein comprises an additional component thatenables efficient delivery of the bacteria to the colon. In someembodiments, pharmaceutical preparation that enables the delivery of thebacteria to the colon can be used. Examples of such formulations includepH sensitive compositions, such as buffered sachet formulations orenteric polymers that release their contents when the pH becomesalkaline after the enteric polymers pass through the stomach. When a pHsensitive composition is used for formulating the pharmaceuticalpreparation, the pH sensitive composition can be a polymer whose pHthreshold of the decomposition of the composition is between about 6.8and about 7.5.

Another embodiment of a pharmaceutical composition useful for deliveryof the bacteria to the colon is one that ensures the delivery to thecolon by delaying the release of the bacteria by approximately 3 to 5hours, which corresponds to the small intestinal transit time. In someembodiments, the pharmaceutical composition for delayed release includesa hydrogel shell. The hydrogel is hydrated and swells upon contact withgastrointestinal fluid, with the result that the contents areeffectively released (released predominantly in the colon). Delayedrelease dosage units include bacteria-containing compositions having amaterial which coats or selectively coats the bacteria. Examples of sucha selective coating material include in vivo degradable polymers,gradually hydrolyzable polymers, gradually water-soluble polymers,and/or enzyme degradable polymers. A wide variety of coating materialsfor efficiently delaying the release is available and includes, forexample, cellulose-based polymers such as hydroxypropyl cellulose,acrylic acid polymers and copolymers such as methacrylic acid polymersand copolymers, and vinyl polymers and copolymers such aspolyvinylpyrrolidone.

Examples of composition enabling the delivery to the colon furtherinclude bioadhesive compositions which specifically adhere to thecolonic mucosal membrane (for example, a polymer described in thespecification of U.S. Pat. No. 6,368,586, hereby incorporated byreference) and compositions into which a protease inhibitor isincorporated for protecting particularly a biopharmaceutical preparationin the gastrointestinal tracts from decomposition due to an activity ofa protease.

An example of a system enabling the delivery to the colon is a system ofdelivering a composition to the colon by pressure change in such a waythat the contents are released by utilizing pressure change caused bygeneration of gas in bacterial fermentation at a distal portion of thestomach. Such a system is not particularly limited, and a more specificexample thereof is a capsule which has contents dispersed in asuppository base and which is coated with a hydrophobic polymer (forexample, ethyl cellulose).

Another example of the system enabling the delivery to the colon is asystem of delivering a composition to the colon, the system beingspecifically decomposed by an enzyme (for example, a carbohydratehydrolase or a carbohydrate reductase) present in the colon. Such asystem is not particularly limited, and more specific examples thereofinclude systems which use food components such as non-starchpolysaccharides, amylose, xanthan gum, and azopolymers.

In some embodiments, Probiotic formulations containing a bacterialstrain listed in Table 1 are provided as encapsulated, enteric coated,or powder forms, with doses ranging up to 10¹¹ cfu (e.g., up to 10¹⁰cfu). In some embodiments, the composition comprises 5×10¹¹ cfu of abacterial strain listed in Table 1 and 10% (w/w) corn starch in acapsule. The capsule is enteric coated for duodenal release at pH5.5 Insome embodiments, the capsule is enteric coated for duodenal release atpH 5.5. In some embodiments, the composition comprises a powder offreeze-dried bacteria of a bacterial strain listed in Table 1 which isdeemed “Qualified Presumption of Safety” (QPS) status. In someembodiments, the composition is stable at frozen or refrigeratedtemperature.

Methods for producing microbial compositions may include three mainprocessing steps. The steps are: organism banking, organism production,and preservation. In certain embodiments, a sample that contains anabundance of the bacterial strain (e.g., a strain of bacteria listed inTable 1) may be cultured by avoiding an isolation step.

For banking, the strains included in the microbial composition may be(1) isolated directly from a specimen or taken from a banked stock, (2)optionally cultured on a nutrient agar or broth that supports growth togenerate viable biomass, and (3) the biomass optionally preserved inmultiple aliquots in long-term storage.

In embodiments using a culturing step, the agar or broth may containnutrients that provide essential elements and specific factors thatenable growth. An example would be a medium composed of 20 g/L glucose,10 g/L yeast extract, 10 g/L soy peptone, 2 g/L citric acid, 1.5 g/Lsodium phosphate monobasic, 100 mg/L ferric ammonium citrate, 80 mg/Lmagnesium sulfate, 10 mg/L hemin chloride, 2 mg/L calcium chloride, 1mg/L menadione. Another example would be a medium composed of 10 g/Lbeef extract, 10 g/L peptone, 5 g/L sodium chloride, 5 g/L dextrose, 3g/L yeast extract, 3 g/L sodium acetate, 1 g/L soluble starch, and 0.5g/L L-cysteine HCl, at pH 6.8. A variety of microbiological media andvariations are well known in the art (e.g., R. M. Atlas, Handbook ofMicrobiological Media (2010) CRC Press). Culture media can be added tothe culture at the start, may be added during the culture, or may beintermittently/continuously flowed through the culture. The strains inthe bacterial composition may be cultivated alone, as a subset of themicrobial composition, or as an entire collection comprising themicrobial composition. As an example, a first strain may be cultivatedtogether with a second strain in a mixed continuous culture, at adilution rate lower than the maximum growth rate of either cell toprevent the culture from washing out of the cultivation.

The inoculated culture is incubated under favorable conditions for atime sufficient to build biomass. For microbial compositions for humanuse this is often at 37° C. temperature, pH, and other parameter withvalues similar to the normal human niche. The environment may beactively controlled, passively controlled (e.g., via buffers), orallowed to drift. For example, for anaerobic bacterial compositions, ananoxic/reducing environment may be employed. This can be accomplished byaddition of reducing agents such as cysteine to the broth, and/orstripping it of oxygen. As an example, a culture of a bacterialcomposition may be grown at 37° C., pH 7, in the medium above,pre-reduced with 1 g/L cysteine-HCl.

When the culture has generated sufficient biomass, it may be preservedfor banking. The organisms may be placed into a chemical milieu thatprotects from freezing (adding ‘cryoprotectants’), drying(‘lyoprotectants’), and/or osmotic shock (‘osmoprotectants’), dispensinginto multiple (optionally identical) containers to create a uniformbank, and then treating the culture for preservation. Containers aregenerally impermeable and have closures that assure isolation from theenvironment. Cryopreservation treatment is accomplished by freezing aliquid at ultra-low temperatures (e.g., at or below −80° C.). Driedpreservation removes water from the culture by evaporation (in the caseof spray drying or ‘cool drying’) or by sublimation (e.g., for freezedrying, spray freeze drying). Removal of water improves long-termmicrobial composition storage stability at temperatures elevated abovecryogenic conditions. If the microbial composition comprises, forexample, spore forming species and results in the production of spores,the final composition may be purified by additional means such asdensity gradient centrifugation. Microbial composition banking may bedone by culturing and preserving the strains individually, or by mixingthe strains together to create a combined bank. As an example ofcryopreservation, a microbial composition culture may be harvested bycentrifugation to pellet the cells from the culture medium, thesupernatant decanted and replaced with fresh culture broth containing15% glycerol. The culture can then be aliquoted into 1 mL cryotubes,sealed, and placed at −80° C. for long-term viability retention. Thisprocedure achieves acceptable viability upon recovery from frozenstorage.

Microbial production may be conducted using similar culture steps tobanking, including medium composition and culture conditions describedabove. It may be conducted at larger scales of operation, especially forclinical development or commercial production. At larger scales, theremay be several subcultivations of the microbial composition prior to thefinal cultivation. At the end of cultivation, the culture is harvestedto enable further formulation into a dosage form for administration.This can involve concentration, removal of undesirable mediumcomponents, and/or introduction into a chemical milieu that preservesthe microbial composition and renders it acceptable for administrationvia the chosen route. For example, a microbial composition may becultivated to a concentration of 10¹⁰ CFU/mL, then concentrated 20-foldby tangential flow microfiltration; the spent medium may be exchanged bydiafiltering with a preservative medium consisting of 2% gelatin, 100 mMtrehalose, and 10 mM sodium phosphate buffer. The suspension can then befreeze-dried to a powder and titrated.

After drying, the powder may be blended to an appropriate potency, andmixed with other cultures and/or a filler such as microcrystallinecellulose for consistency and ease of handling, and the bacterialcomposition formulated as provided herein.

In certain aspects, provided are bacterial compositions foradministration subjects. In some embodiments, the bacterial compositionsare combined with additional active and/or inactive materials in orderto produce a final product, which may be in single dosage unit or in amulti-dose format.

In some embodiments, the composition comprises at least onecarbohydrate. A “carbohydrate” refers to a sugar or polymer of sugars.The terms “saccharide,” “polysaccharide,” “carbohydrate,” and“oligosaccharide” may be used interchangeably. Most carbohydrates arealdehydes or ketones with many hydroxyl groups, usually one on eachcarbon atom of the molecule. Carbohydrates generally have the molecularformula C_(n)H_(2n)O_(n). A carbohydrate may be a monosaccharide, adisaccharide, trisaccharide, oligosaccharide, or polysaccharide. Themost basic carbohydrate is a monosaccharide, such as glucose, sucrose,galactose, mannose, ribose, arabinose, xylose, and fructose.Disaccharides are two joined monosaccharides. Exemplary disaccharidesinclude sucrose, maltose, cellobiose, and lactose. Typically, anoligosaccharide includes between three and six monosaccharide units(e.g., raffinose, stachyose), and polysaccharides include six or moremonosaccharide units. Exemplary polysaccharides include starch,glycogen, and cellulose. Carbohydrates may contain modified saccharideunits such as 2′-deoxyribose wherein a hydroxyl group is removed,2′-fluororibose wherein a hydroxyl group is replaced with a fluorine, orN-acetylglucosamine, a nitrogen-containing form of glucose (e.g.,2′-fluororibose, deoxyribose, and hexose). Carbohydrates may exist inmany different forms, for example, conformers, cyclic forms, acyclicforms, stereoisomers, tautomers, anomers, and isomers.

In some embodiments, the composition comprises at least one lipid. Asused herein, a “lipid” includes fats, oils, triglycerides, cholesterol,phospholipids, fatty acids in any form including free fatty acids. Fats,oils and fatty acids can be saturated, unsaturated (cis or trans) orpartially unsaturated (cis or trans). In some embodiments the lipidcomprises at least one fatty acid selected from lauric acid (12:0),myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16:1),margaric acid (17:0), heptadecenoic acid (17:1), stearic acid (18:0),oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3),octadecatetraenoic acid (18:4), arachidic acid (20:0), eicosenoic acid(20:1), eicosadienoic acid (20:2), eicosatetraenoic acid (20:4),eicosapentaenoic acid (20:5) (EPA), docosanoic acid (22:0), docosenoicacid (22:1), docosapentaenoic acid (22:5), docosahexaenoic acid (22:6)(DHA), and tetracosanoic acid (24:0). In some embodiments, thecomposition comprises at least one modified lipid, for example a lipidthat has been modified by cooking.

In some embodiments, the composition comprises at least one supplementalmineral or mineral source. Examples of minerals include, withoutlimitation: chloride, sodium, calcium, iron, chromium, copper, iodine,zinc, magnesium, manganese, molybdenum, phosphorus, potassium, andselenium. Suitable forms of any of the foregoing minerals includesoluble mineral salts, slightly soluble mineral salts, insoluble mineralsalts, chelated minerals, mineral complexes, non-reactive minerals suchas carbonyl minerals, and reduced minerals, and combinations thereof.

In some embodiments, the composition comprises at least one supplementalvitamin. The at least one vitamin can be fat-soluble or water-solublevitamins. Suitable vitamins include but are not limited to vitamin C,vitamin A, vitamin E, vitamin B12, vitamin K, riboflavin, niacin,vitamin D, vitamin B6, folic acid, pyridoxine, thiamine, pantothenicacid, and biotin. Suitable forms of any of the foregoing are salts ofthe vitamin, derivatives of the vitamin, compounds having the same orsimilar activity of the vitamin, and metabolites of the vitamin.

In some embodiments, the composition comprises an excipient.Non-limiting examples of suitable excipients include a buffering agent,a preservative, a stabilizer, a binder, a compaction agent, a lubricant,a dispersion enhancer, a disintegration agent, a flavoring agent, asweetener, and a coloring agent.

In some embodiments, the excipient is a buffering agent. Non-limitingexamples of suitable buffering agents include sodium citrate, magnesiumcarbonate, magnesium bicarbonate, calcium carbonate, and calciumbicarbonate.

In some embodiments, the excipient comprises a preservative.Non-limiting examples of suitable preservatives include antioxidants,such as alpha-tocopherol and ascorbate, and antimicrobials, such asparabens, chlorobutanol, and phenol.

In some embodiments, the composition comprises a binder as an excipient.Non-limiting examples of suitable binders include starches,pregelatinized starches, gelatin, polyvinylpyrolidone, cellulose,methylcellulose, sodium carboxymethylcellulose, ethylcellulose,polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C₁₂-C₁₈ fattyacid alcohol, polyethylene glycol, polyols, saccharides,oligosaccharides, and combinations thereof.

In some embodiments, the composition comprises a lubricant as anexcipient. Non-limiting examples of suitable lubricants includemagnesium stearate, calcium stearate, zinc stearate, hydrogenatedvegetable oils, sterotex, polyoxyethylene monostearate, talc,polyethyleneglycol, sodium benzoate, sodium lauryl sulfate, magnesiumlauryl sulfate, and light mineral oil.

In some embodiments, the composition comprises a dispersion enhancer asan excipient. Non-limiting examples of suitable dispersants includestarch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin,bentonite, purified wood cellulose, sodium starch glycolate,isoamorphous silicate, and microcrystalline cellulose as high HLBemulsifier surfactants.

In some embodiments, the composition comprises a disintegrant as anexcipient. In some embodiments, the disintegrant is a non-effervescentdisintegrant. Non-limiting examples of suitable non-effervescentdisintegrants include starches such as corn starch, potato starch,pregelatinized and modified starches thereof, sweeteners, clays, such asbentonite, micro-crystalline cellulose, alginates, sodium starchglycolate, gums such as agar, guar, locust bean, karaya, pectin, andtragacanth. In some embodiments, the disintegrant is an effervescentdisintegrant. Non-limiting examples of suitable effervescentdisintegrants include sodium bicarbonate in combination with citricacid, and sodium bicarbonate in combination with tartaric acid.

In some embodiments, the bacterial formulation comprises an entericcoating or micro encapsulation. In certain embodiments, the entericcoating or micro encapsulation improves targeting to a desired region ofthe gastrointestinal tract. For example, in certain embodiments, thebacterial composition comprises an enteric coating and/or microcapsulesthat dissolves at a pH associated with a particular region of thegastrointestinal tract. In some embodiments, the enteric coating and/ormicrocapsules dissolve at a pH of about 5.5-6.2 to release in theduodenum, at a pH value of about 7.2-7.5 to release in the ileum, and/orat a pH value of about 5.6-6.2 to release in the colon. Exemplaryenteric coatings and microcapsules are described, for example, in U.S.Pat. Pub. No. 2016/0022592, which is hereby incorporated by reference inits entirety.

In some embodiments, the composition is a food product (e.g., a food orbeverage) such as a health food or beverage, a food or beverage forinfants, a food or beverage for pregnant women, athletes, seniorcitizens or other specified group, a functional food, a beverage, a foodor beverage for specified health use, a dietary supplement, a food orbeverage for patients, or an animal feed. Specific examples of the foodsand beverages include various beverages such as juices, refreshingbeverages, tea beverages, drink preparations, jelly beverages, andfunctional beverages; alcoholic beverages such as beers;carbohydrate-containing foods such as rice food products, noodles,breads, and pastas; paste products such as fish hams, sausages, pasteproducts of seafood; retort pouch products such as curries, food dressedwith a thick starchy sauces, and Chinese soups; soups; dairy productssuch as milk, dairy beverages, ice creams, cheeses, and yogurts;fermented products such as fermented soybean pastes, yogurts, fermentedbeverages, and pickles; bean products; various confectionery products,including biscuits, cookies, and the like, candies, chewing gums,gummies, cold desserts including jellies, cream caramels, and frozendesserts; instant foods such as instant soups and instant soy-beansoups; microwavable foods; and the like. Further, the examples alsoinclude health foods and beverages prepared in the forms of powders,granules, tablets, capsules, liquids, pastes, and jellies.

In certain embodiments, the bacteria disclosed herein are administeredin conjunction with a prebiotic to the subject. Prebiotics arecarbohydrates which are generally indigestible by a host animal and areselectively fermented or metabolized by bacteria. Prebiotics may beshort-chain carbohydrates (e.g., oligosaccharides) and/or simple sugars(e.g., mono- and di-saccharides) and/or mucins (heavily glycosylatedproteins) that alter the composition or metabolism of a microbiome inthe host. The short chain carbohydrates are also referred to asoligosaccharides, and usually contain from 2 or 3 and up to 8, 9, 10, 15or more sugar moieties. When prebiotics are introduced to a host, theprebiotics affect the bacteria within the host and do not directlyaffect the host. In certain aspects, a prebiotic composition canselectively stimulate the growth and/or activity of one of a limitednumber of bacteria in a host. Prebiotics include oligosaccharides suchas fructooligosaccharides (FOS) (including inulin),galactooligosaccharides (GOS), trans-galactooligosaccharides,xylooligosaccharides (XOS), chitooligosaccharides (COS), soyoligosaccharides (e.g., stachyose and raffinose) gentiooligosaccharides,isomaltooligosaccharides, mannooligosaccharides, maltooligosaccharidesand mannanoligosaccharides. Oligosaccharides are not necessarily singlecomponents, and can be mixtures containing oligosaccharides withdifferent degrees of oligomerization, sometimes including the parentdisaccharide and the monomeric sugars. Various types of oligosaccharidesare found as natural components in many common foods, including fruits,vegetables, milk, and honey. Specific examples of oligosaccharides arelactulose, lactosucrose, palatinose, glycosyl sucrose, guar gum, gumArabic, tagalose, amylose, amylopectin, pectin, xylan, andcyclodextrins. Prebiotics may also be purified or chemically orenzymatically synthesized.

Administration

In certain aspects, provided herein is a method of delivering abacterium and/or a bacterial composition described herein to a subject.In some embodiments of the methods provided herein, the bacteria areadministered in conjunction with the administration of an additionaltherapeutic. In some embodiments, the bacteria is co-formulated in apharmaceutical composition with the additional therapeutic. In someembodiments, the bacteria is co-administered with the additionaltherapeutic. In some embodiments, the additional therapeutic isadministered to the subject before administration of the bacteria (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or55 minutes before, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22 or 23 hours before, or about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days before). In some embodiments,the additional therapeutic is administered to the subject afteradministration of the bacteria (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 minutes after, about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or23 hours after, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14days after). In some embodiments, the same mode of delivery is used todeliver both the bacteria and the additional therapeutic. In someembodiments, different modes of delivery are used to administer thebacteria and the additional therapeutic. For example, in someembodiments, the bacteria is administered orally while the additionaltherapeutic is administered via injection (e.g., an intravenous,intramuscular and/or intratumoral injection).

In certain embodiments, the pharmaceutical compositions, dosage forms,and kits described herein can be administered in conjunction with anyother conventional anti-immune disorder treatment. These treatments maybe applied as necessary and/or as indicated and may occur before,concurrent with or after administration of the pharmaceuticalcompositions, dosage forms, and kits described herein.

The dosage regimen can be any of a variety of methods and amounts, andcan be determined by one skilled in the art according to known clinicalfactors. As is known in the medical arts, dosages for any one patientcan depend on many factors, including the subject's species, size, bodysurface area, age, sex, immunocompetence, and general health, theparticular microorganism to be administered, duration and route ofadministration, the kind and stage of the disease, for example, tumorsize, and other compounds such as drugs being administered concurrently.In addition to the above factors, such levels can be affected by theinfectivity of the microorganism, and the nature of the microorganism,as can be determined by one skilled in the art. In the present methods,appropriate minimum dosage levels of microorganisms can be levelssufficient for the microorganism to survive, grow and replicate. Themethods of treatment described herein may be suitable for the treatmentof an immune disorder (e.g., an autoimmune disease, an inflammatorydisease, an allergy). The dose of the pharmaceutical compositionsdescribed herein may be appropriately set or adjusted in accordance withthe dosage form, the route of administration, the degree or stage of atarget disease, and the like. For example, the general effective dose ofthe agents may range between 0.01 mg/kg body weight/day and 1000 mg/kgbody weight/day, between 0.1 mg/kg body weight/day and 1000 mg/kg bodyweight/day, 0.5 mg/kg body weight/day and 500 mg/kg body weight/day, 1mg/kg body weight/day and 100 mg/kg body weight/day, or between 5 mg/kgbody weight/day and 50 mg/kg body weight/day. The effective dose may be0.01, 0.05, 0.1, 0.5, 1, 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 200, 500, or 1000 mg/kg body weight/day or more, but the dose isnot limited thereto.

In some embodiments, the dose administered to a subject is sufficient toprevent the immune disorder, delay its onset, or slow or stop itsprogression or prevent a relapse of the immune disorder. One skilled inthe art will recognize that dosage will depend upon a variety of factorsincluding the strength of the particular compound employed, as well asthe age, species, condition, and body weight of the subject. The size ofthe dose will also be determined by the route, timing, and frequency ofadministration as well as the existence, nature, and extent of anyadverse side-effects that might accompany the administration of aparticular compound and the desired physiological effect.

Suitable doses and dosage regimens can be determined by conventionalrange-finding techniques known to those of ordinary skill in the art.Generally, treatment is initiated with smaller dosages, which are lessthan the optimum dose of the compound. Thereafter, the dosage isincreased by small increments until the optimum effect under thecircumstances is reached. An effective dosage and treatment protocol canbe determined by routine and conventional means, starting e.g., with alow dose in laboratory animals and then increasing the dosage whilemonitoring the effects, and systematically varying the dosage regimen aswell. Animal studies are commonly used to determine the maximaltolerable dose (“MTD”) of bioactive agent per kilogram weight. Thoseskilled in the art regularly extrapolate doses for efficacy, whileavoiding toxicity, in other species, including humans.

In accordance with the above, in therapeutic applications, the dosagesof the active agents used in accordance with the invention varydepending on the active agent, the age, weight, and clinical conditionof the recipient patient, and the experience and judgment of theclinician or practitioner administering therapy, among other factorsaffecting the selected dosage. Generally, the dose should be sufficientto result in slowing, and preferably regressing, the advancement of animmune disorder.

Separate administrations can include any number of two or moreadministrations (e.g., doses), including two, three, four, five or sixadministrations. One skilled in the art can readily determine the numberof administrations to perform, or the desirability of performing one ormore additional administrations, according to methods known in the artfor monitoring therapeutic methods and other monitoring methods providedherein. In some embodiments, the doses may be separated by at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29 or 30 days or 1, 2, 3, or 4 weeks.Accordingly, the methods provided herein include methods of providing tothe subject one or more administrations of a bacterium, where the numberof administrations can be determined by monitoring the subject, and,based on the results of the monitoring, determining whether or not toprovide one or more additional administrations. Deciding on whether ornot to provide one or more additional administrations can be based on avariety of monitoring results, including, but not limited to, indicationof tumor growth or inhibition of tumor growth, appearance of newmetastases or inhibition of metastasis, the subject's anti-bacteriumantibody titer, the subject's anti-tumor antibody titer, the overallhealth of the subject and/or the weight of the subject.

The time period between administrations can be any of a variety of timeperiods. The time period between administrations can be a function ofany of a variety of factors, including monitoring steps, as described inrelation to the number of administrations, the time period for a subjectto mount an immune response and/or the time period for a subject toclear the bacteria from normal tissue. In one example, the time periodcan be a function of the time period for a subject to mount an immuneresponse; for example, the time period can be more than the time periodfor a subject to mount an immune response, such as more than about oneweek, more than about ten days, more than about two weeks, or more thanabout a month; in another example, the time period can be less than thetime period for a subject to mount an immune response, such as less thanabout one week, less than about ten days, less than about two weeks, orless than about a month. In another example, the time period can be afunction of the time period for a subject to clear the bacteria fromnormal tissue; for example, the time period can be more than the timeperiod for a subject to clear the bacteria from normal tissue, such asmore than about a day, more than about two days, more than about threedays, more than about five days, or more than about a week.

In some embodiments, the delivery of an immune disorder therapeutic incombination with the bacteria described herein reduces the adverseeffects and/or improves the efficacy of the immune disorder therapeutic.

The effective dose of an immune disorder therapeutic described herein isthe amount of therapeutic agent that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, with the least toxicity to the patient. The effectivedosage level can be identified using the methods described herein andwill depend upon a variety of pharmacokinetic factors including theactivity of the particular compositions administered, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts. In general, aneffective dose of an immune disorder therapy will be the amount oftherapeutic agent, which is the lowest dose effective to produce atherapeutic effect. Such an effective dose will generally depend uponthe factors described above.

The toxicity of an immune disorder therapy is the level of adverseeffects experienced by the subject during and following treatment.Adverse events associated with immune disorder therapy toxicity include,but are not limited to, abdominal pain, acid indigestion, acid reflux,allergic reactions, alopecia, anaphylaxis, anemia, anxiety, lack ofappetite, arthralgias, asthenia, ataxia, azotemia, loss of balance, bonepain, bleeding, blood clots, low blood pressure, elevated bloodpressure, difficulty breathing, bronchitis, bruising, low white bloodcell count, low red blood cell count, low platelet count,cardiotoxicity, cystitis, hemorrhagic cystitis, arrhythmias, heart valvedisease, cardiomyopathy, coronary artery disease, cataracts, centralneurotoxicity, cognitive impairment, confusion, conjunctivitis,constipation, coughing, cramping, cystitis, deep vein thrombosis,dehydration, depression, diarrhea, dizziness, dry mouth, dry skin,dyspepsia, dyspnea, edema, electrolyte imbalance, esophagitis, fatigue,loss of fertility, fever, flatulence, flushing, gastric reflux,gastroesophageal reflux disease, genital pain, granulocytopenia,gynecomastia, glaucoma, hair loss, hand-foot syndrome, headache, hearingloss, heart failure, heart palpitations, heartburn, hematoma,hemorrhagic cystitis, hepatotoxicity, hyperamylasemia, hypercalcemia,hyperchloremia, hyperglycemia, hyperkalemia, hyperlipasemia,hypermagnesemia, hypernatremia, hyperphosphatemia, hyperpigmentation,hypertriglyceridemia, hyperuricemia, hypoalbuminemia, hypocalcemia,hypochloremia, hypoglycemia, hypokalemia, hypomagnesemia, hyponatremia,hypophosphatemia, impotence, infection, injection site reactions,insomnia, iron deficiency, itching, joint pain, kidney failure,leukopenia, liver dysfunction, memory loss, menopause, mouth sores,mucositis, muscle pain, myalgias, myelosuppression, myocarditis,neutropenic fever, nausea, nephrotoxicity, neutropenia, nosebleeds,numbness, ototoxicity, pain, palmar-plantar erythrodysesthesia,pancytopenia, pericarditis, peripheral neuropathy, pharyngitis,photophobia, photosensitivity, pneumonia, pneumonitis, proteinuria,pulmonary embolus, pulmonary fibrosis, pulmonary toxicity, rash, rapidheart beat, rectal bleeding, restlessness, rhinitis, seizures, shortnessof breath, sinusitis, thrombocytopenia, tinnitus, urinary tractinfection, vaginal bleeding, vaginal dryness, vertigo, water retention,weakness, weight loss, weight gain, and xerostomia. In general, toxicityis acceptable if the benefits to the subject achieved through therapyoutweigh the adverse events experienced by the subject due to therapy.

In some embodiments, the administration of the bacterial compositiontreats the disease (e.g., cancer, autoimmune disease, inflammatorydisease, metabolic disease).

Therapeutic Agents

In certain aspects, the methods provided herein include theadministration to a subject of a bacterium and/or a bacterialcomposition described herein (e.g., a bacterial composition comprising abacterial strain listed in Table 1) either alone or in combination withanother therapeutic. In some embodiments, the bacterial composition andthe other therapy can be administered to the subject in any order. Insome embodiments, the bacterial composition and the other therapy areadministered conjointly.

In some embodiments the bacterium is administered to the subject beforethe additional therapeutic is administered (e.g., at least 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or24 hours before or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30days before). In some embodiments the bacterium is administered to thesubject after the additional therapeutic is administered (e.g., at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23 or 24 hours after or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29 or 30 days after). In some embodiments, the bacterium and theadditional therapeutic are administered to the subject simultaneously ornearly simultaneously (e.g., administrations occur within an hour ofeach other). In some embodiments, the subject is administered anantibiotic before the bacterium is administered to the subject (e.g., atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23 or 24 hours before or at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29 or 30 days before). In some embodiments, the subject isadministered an antibiotic after the bacterium is administered to thesubject (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours before or at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29 or 30 days after). In some embodiments, thebacterium and the antibiotic are administered to the subjectsimultaneously or nearly simultaneously (e.g., administrations occurwithin an hour of each other).

In certain embodiments, the subject may undergo surgery. Types ofsurgery include but are not limited to preventative, diagnostic orstaging, curative and palliative surgery.

In some embodiments, the additional therapeutic is an antibiotic. Forexample, if the presence of a immune-disorder-associated bacteria and/oran immune-disorder-associated microbiome profile is detected accordingto the methods provided herein, antibiotics can be administered toeliminate the immune-disorder-associated bacteria from the subject.“Antibiotics” broadly refers to compounds capable of inhibiting orpreventing a bacterial infection. Antibiotics can be classified in anumber of ways, including their use for specific infections, theirmechanism of action, their bioavailability, or their spectrum of targetmicrobe (e.g., Gram-negative vs. Gram-positive bacteria, aerobic vs.anaerobic bacteria, etc.) and these may be used to kill specificbacteria in specific areas of the host (“niches”) (Leekha, et al 2011.General Principles of Antimicrobial Therapy. Mayo Clin Proc. 86(2):156-167). In certain embodiments, antibiotics can be used to selectivelytarget bacteria of a specific niche. In some embodiments, antibioticsknown to treat a particular infection that includes an immune disorderniche may be used to target immune-disorder-associated microbes,including immune-disorder-associated bacteria in that niche. In otherembodiments, antibiotics are administered after the bacterial treatment.In some embodiments, antibiotics are administered after the bacterialtreatment to remove the engraftment.

In some aspects, antibiotics can be selected based on their bactericidalor bacteriostatic properties. Bactericidal antibiotics includemechanisms of action that disrupt the cell wall (e.g., β-lactams), thecell membrane (e.g., daptomycin), or bacterial DNA (e.g.,fluoroquinolones). Bacteriostatic agents inhibit bacterial replicationand include sulfonamides, tetracyclines, and macrolides, and act byinhibiting protein synthesis. Furthermore, while some drugs can bebactericidal in certain organisms and bacteriostatic in others, knowingthe target organism allows one skilled in the art to select anantibiotic with the appropriate properties. In certain treatmentconditions, bacteriostatic antibiotics inhibit the activity ofbactericidal antibiotics. Thus, in certain embodiments, bactericidal andbacteriostatic antibiotics are not combined.

Antibiotics include, but are not limited to aminoglycosides, ansamycins,carbacephems, carbapenems, cephalosporins, glycopeptides, lincosamides,lipopeptides, macrolides, monobactams, nitrofurans, oxazolidonones,penicillins, polypeptide antibiotics, quinolones, fluoroquinolone,sulfonamides, tetracyclines, and anti-mycobacterial compounds, andcombinations thereof.

Aminoglycosides include, but are not limited to Amikacin, Gentamicin,Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin, andSpectinomycin. Aminoglycosides are effective, e.g., againstGram-negative bacteria, such as Escherichia coli, Klebsiella,Pseudomonas aeruginosa, and Francisella tularensis, and against certainaerobic bacteria but less effective against obligate/facultativeanaerobes. Aminoglycosides are believed to bind to the bacterial 30S or50S ribosomal subunit thereby inhibiting bacterial protein synthesis.

Ansamycins include, but are not limited to, Geldanamycin, Herbimycin,Rifamycin, and Streptovaricin. Geldanamycin and Herbimycin are believedto inhibit or alter the function of Heat Shock Protein 90.

Carbacephems include, but are not limited to, Loracarbef. Carbacephemsare believed to inhibit bacterial cell wall synthesis.

Carbapenems include, but are not limited to, Ertapenem, Doripenem,Imipenem/Cilastatin, and Meropenem. Carbapenems are bactericidal forboth Gram-positive and Gram-negative bacteria as broad-spectrumantibiotics. Carbapenems are believed to inhibit bacterial cell wallsynthesis.

Cephalosporins include, but are not limited to, Cefadroxil, Cefazolin,Cefalotin, Cefalothin, Cefalexin, Cefaclor, Cefamandole, Cefoxitin,Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone,Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime,Ceftriaxone, Cefepime, Ceftaroline fosamil, and Ceftobiprole. SelectedCephalosporins are effective, e.g., against Gram-negative bacteria andagainst Gram-positive bacteria, including Pseudomonas, certainCephalosporins are effective against methicillin-resistantStaphylococcus aureus (MRSA). Cephalosporins are believed to inhibitbacterial cell wall synthesis by disrupting synthesis of thepeptidoglycan layer of bacterial cell walls.

Glycopeptides include, but are not limited to, Teicoplanin, Vancomycin,and Telavancin. Glycopeptides are effective, e.g., against aerobic andanaerobic Gram-positive bacteria including MRSA and Clostridiumdifficile. Glycopeptides are believed to inhibit bacterial cell wallsynthesis by disrupting synthesis of the peptidoglycan layer ofbacterial cell walls.

Lincosamides include, but are not limited to, Clindamycin andLincomycin. Lincosamides are effective, e.g., against anaerobicbacteria, as well as Staphylococcus, and Streptococcus. Lincosamides arebelieved to bind to the bacterial 50S ribosomal subunit therebyinhibiting bacterial protein synthesis.

Lipopeptides include, but are not limited to, Daptomycin. Lipopeptidesare effective, e.g., against Gram-positive bacteria. Lipopeptides arebelieved to bind to the bacterial membrane and cause rapiddepolarization.

Macrolides include, but are not limited to, Azithromycin,Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin,Troleandomycin, Telithromycin, and Spiramycin. Macrolides are effective,e.g., against Streptococcus and Mycoplasma. Macrolides are believed tobind to the bacterial or 50S ribosomal subunit, thereby inhibitingbacterial protein synthesis.

Monobactams include, but are not limited to, Aztreonam. Monobactams areeffective, e.g., against Gram-negative bacteria. Monobactams arebelieved to inhibit bacterial cell wall synthesis by disruptingsynthesis of the peptidoglycan layer of bacterial cell walls.

Nitrofurans include, but are not limited to, Furazolidone andNitrofurantoin.

Oxazolidonones include, but are not limited to, Linezolid, Posizolid,Radezolid, and Torezolid. Oxazolidonones are believed to be proteinsynthesis inhibitors.

Penicillins include, but are not limited to, Amoxicillin, Ampicillin,Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin,Mezlocillin, Methicillin, Nafcillin, Oxacillin, Penicillin G, PenicillinV, Piperacillin, Temocillin and Ticarcillin. Penicillins are effective,e.g., against Gram-positive bacteria, facultative anaerobes, e.g.,Streptococcus, Borrelia, and Treponema. Penicillins are believed toinhibit bacterial cell wall synthesis by disrupting synthesis of thepeptidoglycan layer of bacterial cell walls.

Penicillin combinations include, but are not limited to,Amoxicillin/clavulanate, Ampicillin/sulbactam, Piperacillin/tazobactam,and Ticarcillin/clavulanate.

Polypeptide antibiotics include, but are not limited to, Bacitracin,Colistin, and Polymyxin B and E. Polypeptide Antibiotics are effective,e.g., against Gram-negative bacteria. Certain polypeptide antibioticsare believed to inhibit isoprenyl pyrophosphate involved in synthesis ofthe peptidoglycan layer of bacterial cell walls, while othersdestabilize the bacterial outer membrane by displacing bacterialcounter-ions.

Quinolones and Fluoroquinolone include, but are not limited to,Ciprofloxacin, Enoxacin, Gatifloxacin, Gemifloxacin, Levofloxacin,Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin,Trovafloxacin, Grepafloxacin, Sparfloxacin, and Temafloxacin.Quinolones/Fluoroquinolone are effective, e.g., against Streptococcusand Neisseria. Quinolones/Fluoroquinolone are believed to inhibit thebacterial DNA gyrase or topoisomerase IV, thereby inhibiting DNAreplication and transcription.

Sulfonamides include, but are not limited to, Mafenide, Sulfacetamide,Sulfadiazine, Silver sulfadiazine, Sulfadimethoxine, Sulfamethizole,Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole,Trimethoprim-Sulfamethoxazole (Co-trimoxazole), andSulfonamidochrysoidine. Sulfonamides are believed to inhibit folatesynthesis by competitive inhibition of dihydropteroate synthetase,thereby inhibiting nucleic acid synthesis.

Tetracyclines include, but are not limited to, Demeclocycline,Doxycycline, Minocycline, Oxytetracycline, and Tetracycline.Tetracyclines are effective, e.g., against Gram-negative bacteria.Tetracyclines are believed to bind to the bacterial 30S ribosomalsubunit thereby inhibiting bacterial protein synthesis.

Anti-mycobacterial compounds include, but are not limited to,Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide,Isoniazid, Pyrazinamide, Rifampicin, Rifabutin, Rifapentine, andStreptomycin.

Suitable antibiotics also include arsphenamine, chloramphenicol,fosfomycin, fusidic acid, metronidazole, mupirocin, platensimycin,quinupristin/dalfopristin, tigecycline, tinidazole, trimethoprimamoxicillin/clavulanate, ampicillin/sulbactam, amphomycin ristocetin,azithromycin, bacitracin, buforin II, carbomycin, cecropin Pl,clarithromycin, erythromycins, furazolidone, fusidic acid, Na fusidate,gramicidin, imipenem, indolicidin, josamycin, magainan II,metronidazole, nitroimidazoles, mikamycin, mutacin B-Ny266, mutacinB-JH1 140, mutacin J-T8, nisin, nisin A, novobiocin, oleandomycin,ostreogrycin, piperacillin/tazobactam, pristinamycin, ramoplanin,ranalexin, reuterin, rifaximin, rosamicin, rosaramicin, spectinomycin,spiramycin, staphylomycin, streptogramin, streptogramin A, synergistin,taurolidine, teicoplanin, telithromycin, ticarcillin/clavulanic acid,triacetyloleandomycin, tylosin, tyrocidin, tyrothricin, vancomycin,vemamycin, and virginiamycin.

In some embodiments, the additional therapeutic is an immunosuppressiveagent, a DMARD, a pain-control drug, a steroid, a non-steroidalantiinflammatory drug (NSAID), or a cytokine antagonist, andcombinations thereof. Representative agents include, but are not limitedto, cyclosporin, retinoids, corticosteroids, propionic acid derivative,acetic acid derivative, enolic acid derivatives, fenamic acidderivatives, Cox-2 inhibitors, lumiracoxib, ibuprophen, cholin magnesiumsalicylate, fenoprofen, salsalate, difunisal, tolmetin, ketoprofen,flurbiprofen, oxaprozin, indomethacin, sulindac, etodolac, ketorolac,nabumetone, naproxen, valdecoxib, etoricoxib, MK0966; rofecoxib,acetominophen, Celecoxib, Diclofenac, tramadol, piroxicam, meloxicam,tenoxicam, droxicam, lornoxicam, isoxicam, mefanamic acid, meclofenamicacid, flufenamic acid, tolfenamic, valdecoxib, parecoxib, etodolac,indomethacin, aspirin, ibuprophen, firocoxib, methotrexate (MTX),antimalarial drugs (e.g., hydroxychloroquine and chloroquine),sulfasalazine, Leflunomide, azathioprine, cyclosporin, gold salts,minocycline, cyclophosphamide, D-penicillamine, minocycline, auranofin,tacrolimus, myocrisin, chlorambucil, TNF alpha antagonists (e.g., TNFalpha antagonists or TNF alpha receptor antagonists), e.g., ADALIMUMAB(Humira®), ETANERCEPT (Enbrel®), INFLIXIMAB (Remicade®; TA-650),CERTOLIZUMAB PEGOL (Cimzia®; CDP870), GOLIMUMAB (Simpom®; CNTO 148),ANAKINRA (Kineret®), RITUXIMAB (Rituxan®; MabThera®), ABATACEPT(Orencia®), TOCILIZUMAB (RoActemra/Actemra®), integrin antagonists(TYSABRI® (natalizumab)), IL-1 antagonists (ACZ885 (Ilaris)), Anakinra(Kineret®)), CD4 antagonists, IL-23 antagonists, IL-20 antagonists, IL-6antagonists, BLyS antagonists (e.g., Atacicept, Benlysta®/LymphoStat-B®(belimumab)), p38 Inhibitors, CD20 antagonists (Ocrelizumab, Ofatumumab(Arzerra®)), interferon gamma antagonists (Fontolizumab), prednisolone,Prednisone, dexamethasone, Cortisol, cortisone, hydrocortisone,methylprednisolone, betamethasone, triamcinolone, beclometasome,fludrocortisone, deoxycorticosterone, aldosterone, Doxycycline,vancomycin, pioglitazone, SBI-087, SCIO-469, Cura-100, Oncoxin+Viusid,TwHF, Methoxsalen, Vitamin D-ergocalciferol, Milnacipran, Paclitaxel,rosig tazone, Tacrolimus (Prograf®), RADOOl, rapamune, rapamycin,fostamatinib, Fentanyl, XOMA 052, Fostamatinib disodium, rosightazone,Curcumin (Longvida™) Rosuvastatin, Maraviroc, ramipnl, Milnacipran,Cobiprostone, somatropin, tgAAC94 gene therapy vector, MK0359, GW856553,esomeprazole, everolimus, trastuzumab, JAK1 and JAK2 inhibitors, pan JAKinhibitors, e.g., tetracyclic pyridone 6 (P6), 325, PF-956980,denosumab, IL-6 antagonists, CD20 antagonistis, CTLA4 antagonists, IL-8antagonists, IL-21 antagonists, IL-22 antagonist, integrin antagonists(Tysarbri® (natalizumab)), VGEF antagnosits, CXCL antagonists, MMPantagonists, defensin antagonists, IL-1 antagonists (including IL-1 betaantagonsits), and IL-23 antagonists (e.g., receptor decoys, antagonisticantibodies, etc.).

In some embodiments, the agent is an immunosuppressive agent. Examplesof immunosuppressive agents include, but are not limited to,corticosteroids, mesalazine, mesalamine, sulfasalazine, sulfasalazinederivatives, immunosuppressive drugs, cyclosporin A, mercaptopurine,azathiopurine, prednisone, methotrexate, antihistamines,glucocorticoids, epinephrine, theophylline, cromolyn sodium,anti-leukotrienes, anti-cholinergic drugs for rhinitis, TLR antagonists,inflammasome inhibitors, anti-cholinergic decongestants, mast-cellstabilizers, monoclonal anti-IgE antibodies, vaccines (e.g., vaccinesused for vaccination where the amount of an allergen is graduallyincreased), cytokine inhibitors, such as anti-IL-6 antibodies, TNFinhibitors such as infliximab, adalimumab, certolizumab pegol,golimumab, or etanercept, and combinations thereof.

In some embodiments, the immune disorder therapy comprises administeringa therapeutic bacteria and/or a therapeutic combination of bacteria tothe subject so a healthy microbiome can be reconstituted in the subject.In some embodiments, therapeutic bacteria is anon-immune-disorder-associated bacteria. In some embodiments therapeuticbacteria is a probiotic bacteria.

In some embodiments, the additional therapeutic is a cancer therapeutic.In some embodiments, the cancer therapeutic is a chemotherapeutic agent.Examples of such chemotherapeutic agents include, but are not limitedto, alkylating agents such as cyclosphosphamide; alkyl sulfonates suchas busulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gammall and calicheamicinomegal1; dynemicin, including dynemicin A; bisphosphonates, such asclodronate; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antibiotic chromophores, aclacinomysins,actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin(including morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharidecomplex); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonicacid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes(especially T-2 toxin, verracurin A, roridin A and anguidine); urethan;vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol;pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide;thiotepa; taxoids, e.g., paclitaxel and doxetaxel; chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinumcoordination complexes such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11);topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO);retinoids such as retinoic acid; capecitabine; and pharmaceuticallyacceptable salts, acids or derivatives of any of the above.

In some embodiments, the cancer therapeutic is a cancer immunotherapyagent. Immunotherapy refers to a treatment that uses a subject's immunesystem to treat cancer, e.g., checkpoint inhibitors, cancer vaccines,cytokines, cell therapy, CAR-T cells, and dendritic cell therapy.Non-limiting examples of immunotherapies are checkpoint inhibitorsinclude Nivolumab (BMS, anti-PD-1), Pembrolizumab (Merck, anti-PD-1),Ipilimumab (BMS, anti-CTLA-4), MEDI4736 (Astra7eneca, anti-PD-L1), andMPDL3280A (Roche, anti-PD-L1). Other immunotherapies may be tumorvaccines, such as Gardail, Cervarix, BCG, sipulencel-T, Gp100:209-217,AGS-003, DCVax-L, Algenpantucel-L, Tergenpantucel-L, TG4010, ProstAtak,Prostvac-V/R-TRICOM, Rindopepimul, E75 peptide acetate, IMA901,POL-103A, Belagenpumatucel-L, GSK1572932A, MDX-1279, GV1001, andTecemotide. Immunotherapy may be administered via injection (e.g.,intravenously, intratumorally, subcutaneously, or into lymph nodes), butmay also be administered orally, topically, or via aerosol.Immunotherapies may comprise adjuvants such as cytokines.

In some embodiments, the immunotherapy agent is an immune checkpointinhibitor. Immune checkpoint inhibition broadly refers to inhibiting thecheckpoints that cancer cells can produce to prevent or downregulate animmune response. Examples of immune checkpoint proteins include, but arenot limited to, CTLA4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA,KIR, LAGS, TIM-3 or VISTA. Immune checkpoint inhibitors can beantibodies or antigen binding fragments thereof that bind to and inhibitan immune checkpoint protein. Examples of immune checkpoint inhibitorsinclude, but are not limited to, nivolumab, pembrolizumab, pidilizumab,AMP-224, AMP-514, STI-A1110, TSR-042, RG-7446, BMS-936559, MEDI-4736,MSB-0020718C, AUR-012 and STI-A1010.

In some embodiments, the immunotherapy agent is an antibody or antigenbinding fragment thereof that, for example, binds to a cancer-associatedantigen. Examples of cancer-associated antigens include, but are notlimited to, adipophilin, AIM-2, ALDH1A1, alpha-actinin-4,alpha-fetoprotein (“AFP”), ARTC1, B-RAF, BAGE-1, BCLX (L), BCR-ABLfusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA,carcinoembryonic antigen (“CEA”), CASP-5, CASP-8, CD274, CD45, Cdc27,CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2,cyclin D1, Cyclin-A1, dek-can fusion protein, DKK1, EFTUD2, Elongationfactor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen(“ETA”), ETV6-AML1 fusion protein, EZH2, FGF5, FLT3-ITD, FN1,G250/MN/CAIX, GAGE-1,2,8, GAGE-3,4,5,6,7, GAS7, glypican-3, GnTV,gp100/Pme117, GPNMB, HAUS3, Hepsin, HER-2/neu, HERV-K-MEL, HLA-A11,HLA-A2, HLA-DOB, hsp70-2, IDO1, IGF2B3, IL13Ralpha2, Intestinal carboxylesterase, K-ras, Kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHN1also known as CCDC110, LAGE-1, LDLR-fucosyltransferaseAS fusion protein,Lengsin, M-CSF, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4,MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, malic enzyme, mammaglobin-A, MART2,MATN, MC1R, MCSP, mdm-2, ME1, Melan-A/MART-1, Meloe, Midkine, MMP-2,MMP-7, MUC1, MUCSAC, mucin, MUM-1, MUM-2, MUM-3, Myosin, Myosin class I,N-raw, NA88-A, neo-PAP, NFYC, NY-BR-1, NY-ESO-1/LAGE-2, OA1, OGT, OS-9,P polypeptide, p53, PAP, PAX5, PBF, pm1-RARalpha fusion protein,polymorphic epithelial mucin (“PEM”), PPP1R3B, PRAME, PRDX5, PSA, PSMA,PTPRK, RAB38/NY-MEL-1, RAGE-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE,secernin 1, SIRT2, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1,survivin, SYT-SSX1 or -SSX2 fusion protein, TAG-1, TAG-2, Telomerase,TGF-betaRII, TPBG, TRAG-3, Triosephosphate isomerase, TRP-1/gp75, TRP-2,TRP2-INT2, tyrosinase, tyrosinase (“TYR”), VEGF, WT1, XAGE-1b/GAGED2a.In some embodiments, the antigen is a neo-antigen.

In some embodiments, the immunotherapy agent is a cancer vaccine and/ora component of a cancer vaccine (e.g., an antigenic peptide and/orprotein). The cancer vaccine can be a protein vaccine, a nucleic acidvaccine or a combination thereof. For example, in some embodiments, thecancer vaccine comprises a polypeptide comprising an epitope of acancer-associated antigen. In some embodiments, the cancer vaccinecomprises a nucleic acid (e.g., DNA or RNA, such as mRNA) that encodesan epitope of a cancer-associated antigen. In some embodiments, thenucleic acid is a vector (e.g., a bacterial vector, viral vector).Examples of bacterial vectors include, but are not limited to,Mycobacterium bovis (BCG), Salmonella Typhimurium ssp., Salmonella Typhissp., Clostridium sp. spores, Escherichia coli Nissle 1917, Escherichiacoli K-12/LLO, Listeria monocytogenes, and Shigella flexneri. Examplesof viral vectors include, but are not limited to, vaccinia, adenovirus,RNA viruses, and replicationdefective avipox, replication-defectivefowlpox, replication-defective canarypox, replicationdefective MVA andreplication-defective adenovirus.

In some embodiments, the cancer immunotherapy comprises administrationof an antigen presenting cell (APC) primed with a cancer-specificantigen. In some embodiments, the APC is a dendritic cell, a macrophageor a B cell.

Examples of cancer-associated antigens include, but are not limited to,adipophilin, AIM-2, ALDH1A1, alpha-actinin-4, alpha-fetoprotein (“AFP”),ARTC1, B-RAF, BAGE-1, BCLX (L), BCR-ABL fusion protein b3a2,beta-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen (“CEA”),CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP,COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin D1, Cyclin-A1, dek-can fusionprotein, DKK1, EFTUD2, Elongation factor 2, ENAH (hMena), Ep-CAM, EpCAM,EphA3, epithelial tumor antigen (“ETA”), ETV6-AML1 fusion protein, EZH2,FGF5, FLT3-ITD, FN1, G250/MN/CAIX, GAGE-1,2,8, GAGE-3,4,5,6,7, GAS7,glypican-3, GnTV, gp100/Pme117, GPNMB, HAUS3, Hepsin, HER-2/neu,HERV-K-MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, IDO1, IGF2B3,IL13Ralpha2, Intestinal carboxyl esterase, K-ras, Kallikrein 4, KIF20A,KK-LC-1, KKLC1, KM-HN-1, KMHN1 also known as CCDC110, LAGE-1,LDLR-fucosyltransferaseAS fusion protein, Lengsin, M-CSF, MAGE-A1,MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9,MAGE-C1, MAGE-C2, malic enzyme, mammaglobin-A, MART2, MATN, MC1R, MCSP,mdm-2, ME1, Melan-A/MART-1, Meloe, Midkine, MMP-2, MMP-7, MUC1, MUC5AC,mucin, MUM-1, MUM-2, MUM-3, Myosin, Myosin class I, N-raw, NA88-A,neo-PAP, NFYC, NY-BR-1, NY-ESO-1/LAGE-2, OA1, OGT, OS-9, P polypeptide,p53, PAP, PAX5, PBF, pm1-RARalpha fusion protein, polymorphic epithelialmucin (“PEM”), PPP1R3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB38/NY-MEL-1,RAGE-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE, secernin 1, SIRT2,SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1, survivin, SYT-SSX1 or-SSX2 fusion protein, TAG-1, TAG-2, Telomerase, TGF-betaRII, TPBG,TRAG-3, Triosephosphate isomerase, TRP-1/gp75, TRP-2, TRP2-INT2,tyrosinase, tyrosinase (“TYR”), VEGF, WT1, XAGE-1b/GAGED2a. In someembodiments, the antigen is a neo-antigen.

In some embodiments, the cancer immunotherapy comprises administrationof a cancer-specific chimeric antigen receptor (CAR). In someembodiments, the CAR is administered on the surface of a T cell. In someembodiments, the CAR binds specifically to a cancer-associated antigen.

In some embodiments, the cancer immunotherapy comprises administrationof a cancer-specific T cell to the subject. In some embodiments, the Tcell is a CD4+ T cell. In some embodiments, the CD4+ T cell is a TH1 Tcell, a TH2 T cell or a TH17 T cell. In some embodiments, the T cellexpresses a T cell receptor specific for a cancer-associated antigen.

In some embodiments, the cancer vaccine is administered with anadjuvant. Examples of adjuvants include, but are not limited to, animmune modulatory protein, Adjuvant 65, α-GalCer, aluminum phosphate,aluminum hydroxide, calcium phosphate, β-Glucan Peptide, CpG ODN DNA,GPI-0100, lipid A, lipopolysaccharide, Lipovant, Montanide,N-acetyl-muramyl-L-alanyl-D-isoglutamine, Pam3CSK4, quil A, choleratoxin (CT) and heat-labile toxin from enterotoxigenic Escherichia coli(LT) including derivatives of these (CTB, mmCT, CTA1-DD, LTB, LTK63,LTR72, dmLT) and trehalose dimycolate.

In some embodiments, the immunotherapy agent is an immune modulatingprotein to the subject. In some embodiments, the immune modulatoryprotein is a cytokine or chemokine. Examples of immune modulatingproteins include, but are not limited to, B lymphocyte chemoattractant(“BLC”), C-C motif chemokine 11 (“Eotaxin-1”), Eosinophil chemotacticprotein 2 (“Eotaxin-2”), Granulocyte colony-stimulating factor(“G-CSF”), Granulocyte macrophage colony-stimulating factor (“GM-CSF”),1-309, Intercellular Adhesion Molecule 1 (“ICAM-1”), Interferon alpha(“IFN-alpha”), Interferon beta (“IFN-beta”) Interferon gamma(“IFN-gamma”), Interlukin-1 alpha (“IL-1 alpha”), Interlukin-1 beta(“IL-1 beta”), Interleukin 1 receptor antagonist (“IL-1 ra”),Interleukin-2 (“IL-2”), Interleukin-4 (“IL-4”), Interleukin-5 (“IL-5”),Interleukin-6 (“IL-6”), Interleukin-6 soluble receptor (“IL-6 sR”),Interleukin-7 (“IL-7”), Interleukin-8 (“IL-8”), Interleukin-10(“IL-10”), Interleukin-11 (“IL-11”), Subunit beta of Interleukin-12(“IL-12 p40” or “IL-12 p70”), Interleukin-13 (“IL-13”), Interleukin-15(“IL-15”), Interleukin-16 (“IL-16”), Interleukin-17A-F (“IL-17A-F”),Interleukin-18 (“IL-18”), Interleukin-21 (“IL-21”), Interleukin-22(“IL-22”), Interleukin-23 (“IL-23”), Interleukin-33 (“IL-33”), Chemokine(C-C motif) Ligand 2 (“MCP-1”), Macrophage colony-stimulating factor(“M-CSF”), Monokine induced by gamma interferon (“MIG”), Chemokine (C-Cmotif) ligand 2 (“MIP-1 alpha”), Chemokine (C-C motif) ligand 4 (“MIP-1beta”), Macrophage inflammatory protein-1-delta (“MIP-1 delta”),Platelet-derived growth factor subunit B (“PDGF-BB”), Chemokine (C-Cmotif) ligand 5, Regulated on Activation, Normal T cell Expressed andSecreted (“RANTES”), TIMP metallopeptidase inhibitor 1 (“TIMP-1”), TIMPmetallopeptidase inhibitor 2 (“TIMP-2”), Tumor necrosis factor,lymphotoxin-alpha (“TNF alpha”), Tumor necrosis factor, lymphotoxin-beta(“TNF beta”), Soluble TNF receptor type 1 (“sTNFRI”), sTNFRIIAR,Brain-derived neurotrophic factor (“BDNF”), Basic fibroblast growthfactor (“bFGF”), Bone morphogenetic protein 4 (“BMP-4”), Bonemorphogenetic protein 5 (“BMP-5”), Bone morphogenetic protein 7(“BMP-7”), Nerve growth factor (“b-NGF”), Epidermal growth factor(“EGF”), Epidermal growth factor receptor (“EGFR”),Endocrine-gland-derived vascular endothelial growth factor (“EG-VEGF”),Fibroblast growth factor 4 (“FGF-4”), Keratinocyte growth factor(“FGF-7”), Growth differentiation factor 15 (“GDF-15”), Glialcell-derived neurotrophic factor (“GDNF”), Growth Hormone,Heparin-binding EGF-like growth factor (“HB-EGF”), Hepatocyte growthfactor (“HGF”), Insulin-like growth factor binding protein 1(“IGFBP-1”), Insulin-like growth factor binding protein 2 (“IGFBP-2”),Insulin-like growth factor binding protein 3 (“IGFBP-3”), Insulin-likegrowth factor binding protein 4 (“IGFBP-4”), Insulin-like growth factorbinding protein 6 (“IGFBP-6”), Insulin-like growth factor 1 (“IGF-1”),Insulin, Macrophage colony-stimulating factor (“M-CSF R”), Nerve growthfactor receptor (“NGF R”), Neurotrophin-3 (“NT-3”), Neurotrophin-4(“NT-4”), Osteoclastogenesis inhibitory factor (“Osteoprotegerin”),Platelet-derived growth factor receptors (“PDGF-AA”),Phosphatidylinositol-glycan biosynthesis (“PIGF”), Skp, Cullin, F-boxcontaining comples (“SCF”), Stem cell factor receptor (“SCF R”),Transforming growth factor alpha (“TGFalpha”), Transforming growthfactor beta-1 (“TGF beta 1”), Transforming growth factor beta-3 (“TGFbeta 3”), Vascular endothelial growth factor (“VEGF”), Vascularendothelial growth factor receptor 2 (“VEGFR2”), Vascular endothelialgrowth factor receptor 3 (“VEGFR3”), VEGF-D 6Ckine, Tyrosine-proteinkinase receptor UFO (“Axl”), Betacellulin (“BTC”), Mucosae-associatedepithelial chemokine (“CCL28”), Chemokine (C-C motif) ligand 27(“CTACK”), Chemokine (C-X-C motif) ligand 16 (“CXCL16”), C-X-C motifchemokine 5 (“ENA-78”), Chemokine (C-C motif) ligand 26 (“Eotaxin-3”),Granulocyte chemotactic protein 2 (“GCP-2”), GRO, Chemokine (C-C motif)ligand 14 (“HCC-1”), Chemokine (C-C motif) ligand 16 (“HCC-4”),Interleukin-9 (“IL-9”), Interleukin-17 F (“IL-17F”),Interleukin-18-binding protein (“IL-18 BPa”), Interleukin-28 A(“IL-28A”), Interleukin 29 (“IL-29”), Interleukin 31 (“IL-31”), C-X-Cmotif chemokine 10 (“IP-10”), Chemokine receptor CXCR3 (“I-TAC”),Leukemia inhibitory factor (“LIF”), Light, Chemokine (C motif) ligand(“Lymphotactin”), Monocyte chemoattractant protein 2 (“MCP-2”), Monocytechemoattractant protein 3 (“MCP-3”), Monocyte chemoattractant protein 4(“MCP-4”), Macrophage-derived chemokine (“MDC”), Macrophage migrationinhibitory factor (“MIF”), Chemokine (C-C motif) ligand 20 (“MIP-3alpha”), C-C motif chemokine 19 (“MIP-3 beta”), Chemokine (C-C motif)ligand 23 (“MPIF-1”), Macrophage stimulating protein alpha chain(“MSPalpha”), Nucleosome assembly protein 1-like 4 (“NAP-2”), Secretedphosphoprotein 1 (“Osteopontin”), Pulmonary and activation-regulatedcytokine (“PARC”), Platelet factor 4 (“PF4”), Stroma cell-derivedfactor-1 alpha (“SDF-1 alpha”), Chemokine (C-C motif) ligand 17(“TARC”), Thymus-expressed chemokine (“TECK”), Thymic stromallymphopoietin (“TSLP 4-IBB”), CD 166 antigen (“ALCAM”), Cluster ofDifferentiation 80 (“B7-1”), Tumor necrosis factor receptor superfamilymember 17 (“BCMA”), Cluster of Differentiation 14 (“CD14”), Cluster ofDifferentiation 30 (“CD30”), Cluster of Differentiation 40 (“CD40Ligand”), Carcinoembryonic antigen-related cell adhesion molecule 1(biliary glycoprotein) (“CEACAM-1”), Death Receptor 6 (“DR6”),Deoxythymidine kinase (“Dtk”), Type 1 membrane glycoprotein(“Endoglin”), Receptor tyrosine-protein kinase erbB-3 (“ErbB3”),Endothelial-leukocyte adhesion molecule 1 (“E-Selectin”), Apoptosisantigen 1 (“Fas”), Fms-like tyrosine kinase 3 (“Flt-3L”), Tumor necrosisfactor receptor superfamily member 1 (“GITR”), Tumor necrosis factorreceptor superfamily member 14 (“HVEM”), Intercellular adhesion molecule3 (“ICAM-3”), IL-1 R4, IL-1 RI, IL-10 Rbeta, IL-17R, IL-2Rgamma, IL-21R,Lysosome membrane protein 2 (“LIMPII”), Neutrophil gelatinase-associatedlipocalin (“Lipocalin-2”), CD62L (“L-Selectin”), Lymphatic endothelium(“LYVE-1”), MHC class I polypeptide-related sequence A (“MICA”), MHCclass I polypeptide-related sequence B (“MICB”), NRG1-beta1, Beta-typeplatelet-derived growth factor receptor (“PDGF Rbeta”), Plateletendothelial cell adhesion molecule (“PECAM-1”), RAGE, Hepatitis A viruscellular receptor 1 (“TIM-1”), Tumor necrosis factor receptorsuperfamily member IOC (“TRAIL R3”), Trappin protein transglutaminasebinding domain (“Trappin-2”), Urokinase receptor (“uPAR”), Vascular celladhesion protein 1 (“VCAM-1”), XEDARActivin A, Agouti-related protein(“AgRP”), Ribonuclease 5 (“Angiogenin”), Angiopoietin 1, Angiostatin,Catheprin S, CD40, Cryptic family protein IB (“Cripto-1”), DAN,Dickkopf-related protein 1 (“DKK-1”), E-Cadherin, Epithelial celladhesion molecule (“EpCAM”), Fas Ligand (FasL or CD95L), Fcg RIIB/C,FoUistatin, Galectin-7, Intercellular adhesion molecule 2 (“ICAM-2”),IL-13 R1, IL-13R2, IL-17B, IL-2 Ra, IL-2 Rb, IL-23, LAP, Neuronal celladhesion molecule (“NrCAM”), Plasminogen activator inhibitor-1(“PAI-1”), Platelet derived growth factor receptors (“PDGF-AB”),Resistin, stromal cell-derived factor 1 (“SDF-1 beta”), sgp130, Secretedfrizzled-related protein 2 (“ShhN”), Sialic acid-bindingimmunoglobulin-type lectins (“Siglec-5”), ST2, Transforming growthfactor-beta 2 (“TGF beta 2”), Tie-2, Thrombopoietin (“TPO”), Tumornecrosis factor receptor superfamily member 10D (“TRAIL R4”), Triggeringreceptor expressed on myeloid cells 1 (“TREM-1”), Vascular endothelialgrowth factor C (“VEGF-C”), VEGFR1Adiponectin, Adipsin (“AND”),Alpha-fetoprotein (“AFP”), Angiopoietin-like 4 (“ANGPTL4”),Beta-2-microglobulin (“B2M”), Basal cell adhesion molecule (“BCAM”),Carbohydrate antigen 125 (“CA125”), Cancer Antigen 15-3 (“CA15-3”),Carcinoembryonic antigen (“CEA”), cAMP receptor protein (“CRP”), HumanEpidermal Growth Factor Receptor 2 (“ErbB2”), Follistatin,Follicle-stimulating hormone (“FSH”), Chemokine (C-X-C motif) ligand 1(“GRO alpha”), human chorionic gonadotropin (“beta HCG”), Insulin-likegrowth factor 1 receptor (“IGF-1 sR”), IL-1 sRII, IL-3, IL-18 Rb, IL-21,Leptin, Matrix metalloproteinase-1 (“MMP-1”), Matrix metalloproteinase-2(“MMP-2”), Matrix metalloproteinase-3 (“MMP-3”), Matrixmetalloproteinase-8 (“MMP-8”), Matrix metalloproteinase-9 (“MMP-9”),Matrix metalloproteinase-10 (“MMP-10”), Matrix metalloproteinase-13(“MMP-13”), Neural Cell Adhesion Molecule (“NCAM-1”), Entactin(“Nidogen-1”), Neuron specific enolase (“NSE”), Oncostatin M (“OSM”),Procalcitonin, Prolactin, Prostate specific antigen (“PSA”), Sialicacid-binding Ig-like lectin 9 (“Siglec-9”), ADAM 17 endopeptidase(“TACE”), Thyroglobulin, Metalloproteinase inhibitor 4 (“TIMP-4”),TSH2B4, Disintegrin and metalloproteinase domain-containing protein 9(“ADAM-9”), Angiopoietin 2, Tumor necrosis factor ligand superfamilymember 13/Acidic leucine-rich nuclear phosphoprotein 32 family member B(“APRIL”), Bone morphogenetic protein 2 (“BMP-2”), Bone morphogeneticprotein 9 (“BMP-9”), Complement component 5a (“C5a”), Cathepsin L,CD200, CD97, Chemerin, Tumor necrosis factor receptor superfamily member6B (“DcR3”), Fatty acid-binding protein 2 (“FABP2”), Fibroblastactivation protein, alpha (“FAP”), Fibroblast growth factor 19(“FGF-19”), Galectin-3, Hepatocyte growth factor receptor (“HGF R”),IFN-gammalpha/beta R2, Insulin-like growth factor 2 (“IGF-2”),Insulin-like growth factor 2 receptor (“IGF-2 R”), Interleukin-1receptor 6 (“IL-1R6”), Interleukin 24 (“IL-24”), Interleukin 33(“IL-33”, Kallikrein 14, Asparaginyl endopeptidase (“Legumain”),Oxidized low-density lipoprotein receptor 1 (“LOX-1”), Mannose-bindinglectin (“MBL”), Neprilysin (“NEP”), Notch homolog 1,translocation-associated (Drosophila) (“Notch-1”), Nephroblastomaoverexpressed (“NOV”), Osteoactivin, Programmed cell death protein 1(“PD-1”), N-acetylmuramoyl-L-alanine amidase (“PGRP-5”), Serpin A4,Secreted frizzled related protein 3 (“sFRP-3”), Thrombomodulin, Tolllikereceptor 2 (“TLR2”), Tumor necrosis factor receptor superfamily member10A (“TRAIL R1”), Transferrin (“TRF”), WIF-1ACE-2, Albumin, AMICA,Angiopoietin 4, B-cell activating factor (“BAFF”), Carbohydrate antigen19-9 (“CA19-9”), CD 163, Clusterin, CRT AM, Chemokine (C-X-C motif)ligand 14 (“CXCL14”), Cystatin C, Decorin (“DCN”), Dickkopf-relatedprotein 3 (“Dkk-3”), Delta-like protein 1 (“DLL1”), Fetuin A,Heparin-binding growth factor 1 (“aFGF”), Folate receptor alpha(“FOLR1”), Furin, GPCR-associated sorting protein 1 (“GASP-1”),GPCR-associated sorting protein 2 (“GASP-2”), Granulocytecolony-stimulating factor receptor (“GCSF R”), Serine protease hepsin(“HAI-2”), Interleukin-17B Receptor (“IL-17B R”), Interleukin 27(“IL-27”), Lymphocyte-activation gene 3 (“LAG-3”), Apolipoprotein A-V(“LDL R”), Pepsinogen I, Retinol binding protein 4 (“RBP4”), SOST,Heparan sulfate proteoglycan (“Syndecan-1”), Tumor necrosis factorreceptor superfamily member 13B (“TACI”), Tissue factor pathwayinhibitor (“TFPI”), TSP-1, Tumor necrosis factor receptor superfamily,member 10b (“TRAIL R2”), TRANCE, Troponin I, Urokinase PlasminogenActivator (“uPA”), Cadherin 5, type 2 or VE-cadherin (vascularendothelial) also known as CD144 (“VE-Cadherin”),WNT1-inducible-signaling pathway protein 1 (“WISP-1”), and ReceptorActivator of Nuclear Factor κB (“RANK”).

In some embodiments, the cancer therapeutic agent is an anti-cancercompound. Exemplary anti-cancer compounds include, but are not limitedto, Alemtuzumab (Campath®), Alitretinoin (Panretin®), Anastrozole(Arimidex®), Bevacizumab (Avastin®), Bexarotene (Targretin®), Bortezomib(Velcade®), Bosutinib (Bosulif®), Brentuximab vedotin (Adcetris®),Cabozantinib (Cometriq™), Carfilzomib (Kyprolis™), Cetuximab (Erbitux®),Crizotinib (Xalkori®), Dasatinib (Sprycel®), Denileukin diftitox(Ontak®), Erlotinib hydrochloride (Tarceva®), Everolimus (Afinitor®),Exemestane (Aromasin®), Fulvestrant (Faslodex®), Gefitinib (Iressa®),Ibritumomab tiuxetan (Zevalin®), Imatinib mesylate (Gleevec®),Ipilimumab (Yervoy™), Lapatinib ditosylate (Tykerb®), Letrozole(Femara®), Nilotinib (Tasigna®), Ofatumumab (Arzerra®), Panitumumab(Vectibix®), Pazopanib hydrochloride (Votrient®), Pertuzumab (Perjeta™),Pralatrexate (Folotyn®), Regorafenib (Stivarga®), Rituximab (Rituxan®),Romidepsin (Istodax®), Sorafenib tosylate (Nexavar®), Sunitinib malate(Sutent®), Tamoxifen, Temsirolimus (Torisel®), Toremifene (Fareston®),Tositumomab and 131I-tositumomab (Bexxar®), Trastuzumab (Herceptin®),Tretinoin (Vesanoid®), Vandetanib (Caprelsa®), Vemurafenib (Zelboraf®),Vorinostat (Zolinza®), and Ziv-aflibercept (Zaltrap®).

Exemplary anti-cancer compounds that modify the function of proteinsthat regulate gene expression and other cellular functions (e.g., HDACinhibitors, retinoid receptor ligants) are Vorinostat (Zolinza®),Bexarotene (Targretin®) and Romidepsin (Istodax®), Alitretinoin(Panretin®), and Tretinoin (Vesanoid®).

Exemplary anti-cancer compounds that induce apoptosis (e.g., proteasomeinhibitors, antifolates) are Bortezomib (Velcade®), Carfilzomib(Kyprolis™), and Pralatrexate (Folotyn®).

Exemplary anti-cancer compounds that increase anti-tumor immune response(e.g., anti CD20, anti CD52; anti-cytotoxic T-lymphocyte-associatedantigen-4) are Rituximab (Rituxan®), Alemtuzumab (Campath®), Ofatumumab(Arzerra®), and Ipilimumab (Yervoy™).

Exemplary anti-cancer compounds that deliver toxic agents to cancercells (e.g., anti-CD20-radionuclide fusions; IL-2-diphtheria toxinfusions; anti-CD30-monomethylauristatin E (MMAE)-fusions) areTositumomab and 131I-tositumomab (Bexxar®) and Ibritumomab tiuxetan(Zevalin®), Denileukin diftitox (Ontak®), and Brentuximab vedotin(Adcetris®).

Other exemplary anti-cancer compounds are small molecule inhibitors andconjugates thereof of, e.g., Janus kinase, ALK, Bcl-2, PARP, PI3K, VEGFreceptor, Braf, MEK, CDK, and HSP90.

Exemplary platinum-based anti-cancer compounds include, for example,cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin,Nedaplatin, Triplatin, and Lipoplatin. Other metal-based drugs suitablefor treatment include, but are not limited to ruthenium-based compounds,ferrocene derivatives, titanium-based compounds, and gallium-basedcompounds.

In some embodiments, the cancer therapeutic is a radioactive moiety thatcomprises a radionuclide. Exemplary radionuclides include, but are notlimited to Cr-51, Cs-131, Ce-134, Se-75, Ru-97, 1-125, Eu-149, Os-189m,Sb-119, I-123, Ho-161, Sb-117, Ce-139, In-111, Rh-103m, Ga-67, T1-201,Pd-103, Au-195, Hg-197, Sr-87m, Pt-191, P-33, Er-169, Ru-103, Yb-169,Au-199, Sn-121, Tm-167, Yb-175, In-113m, Sn-113, Lu-177, Rh-105,Sn-117m, Cu-67, Sc-47, Pt-195m, Ce-141, 1-131, Tb-161, As-77, Pt-197,Sm-153, Gd-159, Tm-173, Pr-143, Au-198, Tm-170, Re-186, Ag-111, Pd-109,Ga-73, Dy-165, Pm-149, Sn-123, Sr-89, Ho-166, P-32, Re-188, Pr-142,Ir-194, In-114m/In-114, and Y-90.

In some embodiments, the cancer therapeutic is an antibiotic. Forexample, if the presence of a cancer-associated bacteria and/or acancer-associated microbiome profile is detected according to themethods provided herein, antibiotics can be administered to eliminatethe cancer-associated bacteria from the subject. “Antibiotics” broadlyrefers to compounds capable of inhibiting or preventing a bacterialinfection. Antibiotics can be classified in a number of ways, includingtheir use for specific infections, their mechanism of action, theirbioavailability, or their spectrum of target microbe (e.g.,Gram-negative vs. Gram-positive bacteria, aerobic vs. anaerobicbacteria, etc.) and these may be used to kill specific bacteria inspecific areas of the host (“niches”) (Leekha, et al 2011. GeneralPrinciples of Antimicrobial Therapy. Mayo Clin Proc. 86(2): 156-167). Incertain embodiments, antibiotics can be used to selectively targetbacteria of a specific niche. In some embodiments, antibiotics known totreat a particular infection that includes a cancer niche may be used totarget cancer-associated microbes, including cancer-associated bacteriain that niche. In other embodiments, antibiotics are administered afterthe bacterial treatment. In some embodiments, antibiotics areadministered after the bacterial treatment to remove the engraftment.

Immune Disorders

In some embodiments, the methods and compositions described hereinrelate to the treatment or prevention of a disease or disorderassociated with a pathological immune response, such as an autoimmunedisease, an allergic reaction and/or an inflammatory disease. In someembodiments, the disease or disorder is an inflammatory bowel disease(e.g., Crohn's disease or ulcerative colitis). In some embodiments, themethods and compositions described herein relate to the treatment orprevention of delayed-type hypersensitivity, autoimmune myocarditis,granulomas, peripheral neuropathies, Hashimoto's thyroiditis,inflammation of the colon, colitis, microscopic colitis, collagenouscolitis, diversion colitis, chemical colitis, ischemic colitis,indeterminate colitis, atypical colitis.

The methods described herein can be used to treat any subject in needthereof. As used herein, a “subject in need thereof” includes anysubject that has a disease or disorder associated with a pathologicalimmune response (e.g., an inflammatory bowel disease), as well as anysubject with an increased likelihood of acquiring a such a disease ordisorder.

The compositions described herein can be used, for example, as apharmaceutical composition for preventing or treating (reducing,partially or completely, the adverse effects of) an autoimmune disease,such as chronic inflammatory bowel disease, systemic lupuserythematosus, psoriasis, muckle-wells syndrome, rheumatoid arthritis,multiple sclerosis, or Hashimoto's disease; an allergic disease, such asa food allergy, pollenosis, or asthma; an infectious disease, such as aninfection with Clostridium difficile; an inflammatory disease such as aTNF-mediated inflammatory disease (e.g., an inflammatory disease of thegastrointestinal tract, such as pouchitis, a cardiovascular inflammatorycondition, such as atherosclerosis, or an inflammatory lung disease,such as chronic obstructive pulmonary disease); a pharmaceuticalcomposition for suppressing rejection in organ transplantation or othersituations in which tissue rejection might occur; a supplement, food, orbeverage for improving immune functions; or a reagent for suppressingthe proliferation or function of immune cells.

In some embodiments, the methods provided herein are useful for thetreatment of inflammation. In certain embodiments, the inflammation ofany tissue and organs of the body, including musculoskeletalinflammation, vascular inflammation, neural inflammation, digestivesystem inflammation, ocular inflammation, inflammation of thereproductive system, and other inflammation, as discussed below.

Immune disorders of the musculoskeletal system include, but are notlimited, to those conditions affecting skeletal joints, including jointsof the hand, wrist, elbow, shoulder, jaw, spine, neck, hip, knew, ankle,and foot, and conditions affecting tissues connecting muscles to bonessuch as tendons. Examples of such immune disorders, which may be treatedwith the methods and compositions described herein include, but are notlimited to, arthritis (including, for example, osteoarthritis,rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, acuteand chronic infectious arthritis, arthritis associated with gout andpseudogout, and juvenile idiopathic arthritis), tendonitis, synovitis,tenosynovitis, bursitis, fibrositis (fibromyalgia), epicondylitis,myositis, and osteitis (including, for example, Paget's disease,osteitis pubis, and osteitis fibrosa cystic).

Ocular immune disorders refers to an immune disorder that affects anystructure of the eye, including the eye lids. Examples of ocular immunedisorders which may be treated with the methods and compositionsdescribed herein include, but are not limited to, blepharitis,blepharochalasis, conjunctivitis, dacryoadenitis, keratitis,keratoconjunctivitis sicca (dry eye), scleritis, trichiasis, and uveitis

Examples of nervous system immune disorders which may be treated withthe methods and compositions described herein include, but are notlimited to, encephalitis, Guillain-Barre syndrome, meningitis,neuromyotonia, narcolepsy, multiple sclerosis, myelitis andschizophrenia. Examples of inflammation of the vasculature or lymphaticsystem which may be treated with the methods and compositions describedherein include, but are not limited to, arthrosclerosis, arthritis,phlebitis, vasculitis, and lymphangitis.

Examples of digestive system immune disorders which may be treated withthe methods and compositions described herein include, but are notlimited to, cholangitis, cholecystitis, enteritis, enterocolitis,gastritis, gastroenteritis, inflammatory bowel disease, ileitis, andproctitis. Inflammatory bowel diseases include, for example, certainart-recognized forms of a group of related conditions. Several majorforms of inflammatory bowel diseases are known, with Crohn's disease(regional bowel disease, e.g., inactive and active forms) and ulcerativecolitis (e.g., inactive and active forms) the most common of thesedisorders. In addition, the inflammatory bowel disease encompassesirritable bowel syndrome, microscopic colitis, lymphocytic-plasmocyticenteritis, coeliac disease, collagenous colitis, lymphocytic colitis andeosinophilic enterocolitis. Other less common forms of IBD includeindeterminate colitis, pseudomembranous colitis (necrotizing colitis),ischemic inflammatory bowel disease, Behcet's disease, sarcoidosis,scleroderma, IBD-associated dysplasia, dysplasia associated masses orlesions, and primary sclerosing cholangitis.

Examples of reproductive system immune disorders which may be treatedwith the methods and compositions described herein include, but are notlimited to, cervicitis, chorioamnionitis, endometritis, epididymitis,omphalitis, oophoritis, orchitis, salpingitis, tubo-ovarian abscess,urethritis, vaginitis, vulvitis, and vulvodynia.

The methods and compositions described herein may be used to treatautoimmune conditions having an inflammatory component. Such conditionsinclude, but are not limited to, acute disseminated alopeciauniversalise, Behcet's disease, Chagas' disease, chronic fatiguesyndrome, dysautonomia, encephalomyelitis, ankylosing spondylitis,aplastic anemia, hidradenitis suppurativa, autoimmune hepatitis,autoimmune oophoritis, celiac disease, Crohn's disease, diabetesmellitus type 1, giant cell arteritis, goodpasture's syndrome, Grave'sdisease, Guillain-Barre syndrome, Hashimoto's disease, Henoch-Schonleinpurpura, Kawasaki's disease, lupus erythematosus, microscopic colitis,microscopic polyarteritis, mixed connective tissue disease, Muckle-Wellssyndrome, multiple sclerosis, myasthenia gravis, opsoclonus myoclonussyndrome, optic neuritis, ord's thyroiditis, pemphigus, polyarteritisnodosa, polymyalgia, rheumatoid arthritis, Reiter's syndrome, Sjogren'ssyndrome, temporal arteritis, Wegener's granulomatosis, warm autoimmunehaemolytic anemia, interstitial cystitis, Lyme disease, morphea,psoriasis, sarcoidosis, scleroderma, ulcerative colitis, and vitiligo.

The methods and compositions described herein may be used to treatT-cell mediated hypersensitivity diseases having an inflammatorycomponent. Such conditions include, but are not limited to, contacthypersensitivity, contact dermatitis (including that due to poison ivy),uticaria, skin allergies, respiratory allergies (hay fever, allergicrhinitis, house dustmite allergy) and gluten-sensitive enteropathy(Celiac disease).

Other immune disorders which may be treated with the methods andcompositions include, for example, appendicitis, dermatitis,dermatomyositis, endocarditis, fibrositis, gingivitis, glossitis,hepatitis, hidradenitis suppurativa, iritis, laryngitis, mastitis,myocarditis, nephritis, otitis, pancreatitis, parotitis, percarditis,peritonoitis, pharyngitis, pleuritis, pneumonitis, prostatistis,pyelonephritis, and stomatisi, transplant rejection (involving organssuch as kidney, liver, heart, lung, pancreas (e.g., islet cells), bonemarrow, cornea, small bowel, skin allografts, skin homografts, and heartvalve xengrafts, sewrum sickness, and graft vs host disease), acutepancreatitis, chronic pancreatitis, acute respiratory distress syndrome,Sexary's syndrome, congenital adrenal hyperplasis, nonsuppurativethyroiditis, hypercalcemia associated with cancer, pemphigus, bullousdermatitis herpetiformis, severe erythema multiforme, exfoliativedermatitis, seborrheic dermatitis, seasonal or perennial allergicrhinitis, bronchial asthma, contact dermatitis, atopic dermatitis, drughypersensistivity reactions, allergic conjunctivitis, keratitis, herpeszoster ophthalmicus, iritis and oiridocyclitis, chorioretinitis, opticneuritis, symptomatic sarcoidosis, fulminating or disseminated pulmonarytuberculosis chemotherapy, idiopathic thrombocytopenic purpura inadults, secondary thrombocytopenia in adults, acquired (autoimmune)haemolytic anemia, leukaemia and lymphomas in adults, acute leukaemia ofchildhood, regional enteritis, autoimmune vasculitis, multiplesclerosis, chronic obstructive pulmonary disease, solid organ transplantrejection, sepsis. Preferred treatments include treatment of transplantrejection, rheumatoid arthritis, psoriatic arthritis, multiplesclerosis, Type 1 diabetes, asthma, inflammatory bowel disease, systemiclupus erythematosus, psoriasis, chronic obstructive pulmonary disease,and inflammation accompanying infectious conditions (e.g., sepsis).

The methods and compositions described herein may be used to treatmetabolic disorders and metabolic syndromes. Such conditions include,but are not limited to, Type II Diabetes, Encephalopathy, Tay-Sachsdisease, Krabbe disease, Galactosemia, Phenylketonuria (PKU), and Maplesyrup urine disease.

The methods and compositions described herein may be used to treatneurodegenerative and neurological diseases. Such conditions include,but are not limited to, Parkinson's disease, Alzheimer's disease, priondisease, Huntington's disease, motor neurone diseases (MND),spinocerebellar ataxia, spinal muscular atrophy, dystonia,idiopathicintracranial hypertension, epilepsy, nervous system disease,central nervous system disease, movement disorders, multiple sclerosis,encephalopathy, peripheral neuropathy and post-operative cognitivedysfunction.

Cancer

In some embodiments, the methods and compositions described hereinrelate to the treatment of cancer. In some embodiments, any cancer canbe treated using the methods described herein. Examples of cancers thatmay treated by methods and compositions described herein include, butare not limited to, cancer cells from the bladder, blood, bone, bonemarrow, brain, breast, colon, esophagus, gastrointestine, gum, head,kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach,testis, tongue, or uterus. In addition, the cancer may specifically beof the following histological type, though it is not limited to these:neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant andspindle cell carcinoma; small cell carcinoma; papillary carcinoma;squamous cell carcinoma; lymphoepithelial carcinoma; basal cellcarcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillarytransitional cell carcinoma; adenocarcinoma; gastrinoma, malignant;cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellularcarcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoidcystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma,familial polyposis coli; solid carcinoma; carcinoid tumor, malignant;branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma;chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma;basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma;follicular adenocarcinoma; papillary and follicular adenocarcinoma;nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma;endometroid carcinoma; skin appendage carcinoma; apocrineadenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma;mucoepidermoid carcinoma; cystadenocarcinoma; papillarycystadenocarcinoma; papillary serous cystadenocarcinoma; mucinouscystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma;infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma;inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma;adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma,malignant; ovarian stromal tumor, malignant; thecoma, malignant;granulosa cell tumor, malignant; and roblastoma, malignant; sertoli cellcarcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant;paraganglioma, malignant; extra-mammary paraganglioma, malignant;pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanoticmelanoma; superficial spreading melanoma; malig melanoma in giantpigmented nevus; epithelioid cell melanoma; blue nevus, malignant;sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma;liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonalrhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixedtumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma;carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant;phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant;dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii,malignant; choriocarcinoma; mesonephroma, malignant; hemangio sarcoma;hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma,malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma;chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma;giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant;ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblasticfibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant;ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillaryastrocytoma; astroblastoma; glioblastoma; oligodendroglioma;oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma;ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactoryneurogenic tumor; meningioma, malignant; neurofibrosarcoma;neurilemmoma, malignant; granular cell tumor, malignant; malignantlymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma;malignant lymphoma, small lymphocytic; malignant lymphoma, large cell,diffuse; malignant lymphoma, follicular; mycosis fungoides; otherspecified non-Hodgkin's lymphomas; malignant histiocytosis; multiplemyeloma; mast cell sarcoma; immunoproliferative small intestinaldisease; leukemia; lymphoid leukemia; plasma cell leukemia;erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia;basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mastcell leukemia; megakaryoblastic leukemia; myeloid sarcoma; plasmacytoma,colorectal cancer, rectal cancer, and hairy cell leukemia.

In some embodiments, the methods and compositions provided herein relateto the treatment of a leukemia. The term “leukemia” is meant broadlyprogressive, malignant diseases of the hematopoietic organs/systems andis generally characterized by a distorted proliferation and developmentof leukocytes and their precursors in the blood and bone marrow.Non-limiting examples of leukemia diseases include, acute nonlymphocyticleukemia, chronic lymphocytic leukemia, acute granulocytic leukemia,chronic granulocytic leukemia, acute promyelocytic leukemia, adultT-cell leukemia, aleukemic leukemia, a leukocythemic leukemia,basophilic leukemia, blast cell leukemia, bovine leukemia, chronicmyelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilicleukemia, Gross' leukemia, Rieder cell leukemia, Schilling's leukemia,stem cell leukemia, subleukemic leukemia, undifferentiated cellleukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblasticleukemia, histiocytic leukemia, stem cell leukemia, acute monocyticleukemia, leukopenic leukemia, lymphatic leukemia, lymphoblasticleukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoidleukemia, lymphosarcoma cell leukemia, mast cell leukemia,megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia,myeloblastic leukemia, myelocytic leukemia, myeloid granulocyticleukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cellleukemia, plasmacytic leukemia, and promyelocytic leukemia.

In some embodiments, the methods and compositions provided herein relateto the treatment of a carcinoma. The term “carcinoma” refers to amalignant growth made up of epithelial cells tending to infiltrate thesurrounding tissues, and/or resist physiological and non-physiologicalcell death signals and gives rise to metastases. Non-limiting exemplarytypes of carcinomas include, acinar carcinoma, acinous carcinoma,adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum,carcinoma of adrenal cortex, alveolar carcinoma, alveolar cellcarcinoma, basal cell carcinoma, carcinoma basocellulare, basaloidcarcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma,bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma,cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma,comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma encuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cellcarcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma,encephaloid carcinoma, epiennoid carcinoma, carcinoma epithelialeadenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum,gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma,signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma,solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma,carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma,string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes,transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma,verrucous carcinoma, carcinoma villosum, carcinoma gigantocellulare,glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma,hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma,hyaline carcinoma, hypernephroid carcinoma, infantile embryonalcarcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelialcarcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cellcarcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatouscarcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullarycarcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma,carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma,carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes,naspharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans,osteoid carcinoma, papillary carcinoma, periportal carcinoma,preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma,renal cell carcinoma of kidney, reserve cell carcinoma, carcinomasarcomatodes, schneiderian carcinoma, scirrhous carcinoma, merkel cellcarcinoma, salivary gland carcinoma and carcinoma scroti.

In some embodiments, the methods and compositions provided herein relateto the treatment of a sarcoma. The term “sarcoma” generally refers to atumor which is made up of a substance like the embryonic connectivetissue and is generally composed of closely packed cells embedded in afibrillar, heterogeneous, or homogeneous substance. Sarcomas include,but are not limited to, chondrosarcoma, fibrosarcoma, lymphosarcoma,melanosarcoma, myxosarcoma, osteosarcoma, endometrial sarcoma, stromalsarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giantcell sarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolarsoft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloromasarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma,granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmentedhemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma,immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma,Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymomasarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma,serocystic sarcoma, synovial sarcoma, and telangiectaltic sarcoma.

Additional exemplary neoplasias that can be treated using the methodsand compositions described herein include Hodgkin's Disease,Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer,ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis,primary macroglobulinemia, small-cell lung tumors, primary brain tumors,stomach cancer, colon cancer, malignant pancreatic insulanoma, malignantcarcinoid, premalignant skin lesions, testicular cancer, lymphomas,thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tractcancer, malignant hypercalcemia, cervical cancer, endometrial cancer,and adrenal cortical cancer.

In some embodiments, the cancer treated is a melanoma. The term“melanoma” is taken to mean a tumor arising from the melanocytic systemof the skin and other organs. Non-limiting examples of melanomas areHarding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma,malignant melanoma, acral-lentiginous melanoma, amelanotic melanoma,benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, nodularmelanoma subungal melanoma, and superficial spreading melanoma.

Particular categories of tumors that can be treated using methods andcompositions described herein include lymphoproliferative disorders,breast cancer, ovarian cancer, prostate cancer, cervical cancer,endometrial cancer, bone cancer, liver cancer, stomach cancer, coloncancer, pancreatic cancer, cancer of the thyroid, head and neck cancer,cancer of the central nervous system, cancer of the peripheral nervoussystem, skin cancer, kidney cancer, as well as metastases of all theabove. Particular types of tumors include hepatocellular carcinoma,hepatoma, hepatoblastoma, rhabdomyosarcoma, esophageal carcinoma,thyroid carcinoma, ganglioblastoma, fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, Ewing's tumor, leimyosarcoma, rhabdotheliosarcoma,invasive ductal carcinoma, papillary adenocarcinoma, melanoma, pulmonarysquamous cell carcinoma, basal cell carcinoma, adenocarcinoma (welldifferentiated, moderately differentiated, poorly differentiated orundifferentiated), bronchioloalveolar carcinoma, renal cell carcinoma,hypernephroma, hypernephroid adenocarcinoma, bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testiculartumor, lung carcinoma including small cell, non-small and large celllung carcinoma, bladder carcinoma, glioma, astrocyoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, retinoblastoma, neuroblastoma,colon carcinoma, rectal carcinoma, hematopoietic malignancies includingall types of leukemia and lymphoma including: acute myelogenousleukemia, acute myelocytic leukemia, acute lymphocytic leukemia, chronicmyelogenous leukemia, chronic lymphocytic leukemia, mast cell leukemia,multiple myeloma, myeloid lymphoma, Hodgkin's lymphoma, non-Hodgkin'slymphoma.

Cancers treated in certain embodiments also include precancerouslesions, e.g., actinic keratosis (solar keratosis), moles (dysplasticnevi), acitinic chelitis (farmer's lip), cutaneous horns, Barrett'sesophagus, atrophic gastritis, dyskeratosis congenita, sideropenicdysphagia, lichen planus, oral submucous fibrosis, actinic (solar)elastosis and cervical dysplasia.

Cancers treated in some embodiments include non-cancerous or benigntumors, e.g., of endodermal, ectodermal or mesenchymal origin,including, but not limited to cholangioma, colonic polyp, adenoma,papilloma, cystadenoma, liver cell adenoma, hydatidiform mole, renaltubular adenoma, squamous cell papilloma, gastric polyp, hemangioma,osteoma, chondroma, lipoma, fibroma, lymphangioma, leiomyoma,rhabdomyoma, astrocytoma, nevus, meningioma, and ganglioneuroma.

EXAMPLES Example 1: Immune Modulation of Human Commensal Bacteria in aKLH-Based Delayed Type Hypersensitivity Model

Delayed-type hypersensitivity (DTH) is an animal model of atopicdermatitis (or allergic contact dermatitis), as reviewed by Petersen etal. (In vivo pharmacological disease models for psoriasis and atopicdermatitis in drug discovery. Basic & Clinical Pharm & Toxicology. 2006.99(2): 104-115; see also Irving C. Allen (ed.) Mouse Models of InnateImmunity: Methods and Protocols, Methods in Molecular Biology, 2013.vol. 1031, DOI 10.1007/978-1-62703-481-4_13). It can be induced in avariety of mouse and rat strains using various haptens or antigens, forexample using an antigen emulsified with an adjuvant. DTH ischaracterized by sensitization as well as an antigen-specific Tcell-mediated reaction that results in erythema, edema, and cellularinfiltration—especially infiltration of antigen presenting cells (APCs),eosinophils, activated CD4+ T cells, and cytokine-expressing Th2 cells.

The test formulations were prepared for KLH-based delayed typehypersensitivity model. The DTH model provides an in vivo mechanism tostudy the cell-mediated immune response, and resulting inflammation,following exposure to a specific antigen to which the mice have beensensitized. Several variations of the DTH model have been used and arewell known in the art (Irving C. Allen (ed.). Mouse Models of InnateImmunity: Methods and Protocols, Methods in Molecular Biology. Vol.1031, DOI 10.1007/978-1-62703-481-4_13, Springer Science+Business Media,LLC 2013). For example, the emulsion of Keyhole Limpet Hemocyanin (KLH)and Complete Freund's Adjuvant (CFA) are prepared freshly on the day ofimmunization (day 0). To this end, 8 mg of KLH powder is weighed and isthoroughly re-suspended in 16 mL saline. An emulsion is prepared bymixing the KLH/saline with an equal volume of CFA solution (e.g. 10 mLKLH/saline+10 mL CFA solution) using syringes and a luer lock connector.KLH and CFA is mixed vigorously for several minutes to form awhite-colored emulsion to obtain maximum stability. A drop test isperformed to check if a homogenous emulsion is obtained, mixing iscontinued until an intact drop remains visible in the water.

On day 0, C57Bl/6J female mice, approximately 7 weeks old, were primedwith KLH antigen in CFA by subcutaneous immunization (4 sites, 50 μL persite).

Dexamethasone, a corticosteroid, is a known anti-inflammatory thatameliorates DTH reactions in mice, and serves as a positive control forsuppressing inflammation in this model (Taube and Carlsten, Action ofdexamethasone in the suppression of delayed-type hypersensitivity inreconstituted SCID mice. Inflamm Res. 2000. 49(10): 548-52). For thepositive control group, a stock solution of 17 mg/mL of Dexamethasonewas prepared on Day 0 by diluting 6.8 mg Dexamethasone in 400 μL 96%ethanol. For each day of dosing, a working solution is prepared bydiluting the stock solution 100× in sterile PBS to obtain a finalconcentration of 0.17 mg/mL in a septum vial for intraperitoneal dosing.Dexamethasone-treated mice received 100 μL Dexamethasone i.p. (5 mL/kgof a 0.17 mg/mL solution). Frozen sucrose served as the negative control(vehicle). Veillonella Strains A, B, and C were dosed at 1×10{circumflexover ( )}10 CFU p.o. daily. Dexamethasone (positive control), vehicle(negative control), and Bifidobacterium animalis lactis (10 mg powder)were dosed daily.

On day 8, mice were challenged intradermally (i.d.) with 10 μg KLH insaline (in a volume of 10 μL) in the left ear Inflammatory responseswere measured using methods known in the art. Ear pinna thickness wasmeasured at 24 hours following antigen challenge (FIG. 1). As determinedby ear thickness, Veillonella Strains A, B, and C were efficacious atsuppressing inflammation compared to mice that received vehicle alone(comparable to Dexamethasone treatment).

The efficacy of Veillonella strains may be studied further using variedtiming and varied doses. For instance, treatment with a Veillonellabacterial composition may be initiated at some point, either around thetime of priming or around the time of DTH challenge. For example,Veillonella (1×10⁹ CFU per mouse per day) may be administered at thesame time as the subcutaneous injections (day 0), or administered priorto, or upon, intradermal injection. Veillonella strains (e.g. Strain A,Strain B, or Strain C) may be administered at varied doses and atdefined intervals, and in various combinations. For example, some miceare intravenously injected with Veillonella Strain A at a range ofbetween 1×10⁴ and 5×10⁹ bacterial cells per mouse. Some mice receive amixture of Strain A and/or Strain B and/or Strain C. While some micereceive a Veillonella strain through i.v. injection, other mice mayreceive a Veillonella strain through intraperitoneal (i.p.) injection,subcutaneous (s.c.) injection, nasal route administration, oral gavage,topical administration, intradermal (i.d.) injection, or other means ofadministration. Some mice may receive a Veillonella strain every day(e.g. starting on day 0), while others may receive a Veillonella strainat alternative intervals (e.g. every other day, or once every threedays). The bacterial cells may be live, dead, or weakened. The bacterialcells may be harvested fresh (or frozen) and administered, or they maybe irradiated or heat-killed prior to administration.

For example, mice were sensitized to KLH as described above, and groupsreceived live or irradiated Veillonella (25 kGy). Mice received vehicle,Dexamethasone, viable Veillonella Strain B (5.09×10{circumflex over( )}9), irradiated Veillonella Strain B (5.09×10{circumflex over ( )}9,25 kGy), viable Veillonella Strain C (5.38×10{circumflex over ( )}9), orirradiated Veillonella Strain C (5.38×10{circumflex over ( )}9, 25 kGy).Mice were dosed on days 1-9, and challenged on day 8 with earmeasurements taken on day 9 (24 hours) and day 10 (48 hours). Both liveand irradiated Veillonella Strain B are efficacious in reducing earswelling at both 24 and 48 hours compared to vehicle (negative control)(FIG. 2 and FIG. 3, respectively). Irradiated Veillonella Strain B wasmore efficacious than non-irradiated Strain B, and irradiatedVeillonella Strain B was even more efficacious than Dexamethasone atinhibiting ear swelling. Both viable and irradiated Veillonella Strain Cgroups demonstrated efficacy at 24 and 48 timepoints In contrast toStrain B, the irradiation of Veillonella Strain C neither enhanced nordiminished its efficacy.

Alternative cell dosing ranges and/or radiation dosing may be studied.For example, some groups of mice may receive between 1×10⁴ and 5×10⁹bacterial cells in an administration. Alternatively, some bacterialcells may be irradiated at higher or lower radiation doses, for examplebetween 15 kGy or 35 kGy. Bacterial cell composition administration maybe varied by route of administration, dose, and schedule. This caninclude oral gavage, i.v. injection, i.p. injection, i.d. injection,topical administration, or nasal route administration.

Some groups of mice may be treated with anti-inflammatory agent(s) (e.g.anti-CD154, blockade of members of the TNF family, or other treatment),and/or an appropriate control (e.g. vehicle or control antibody) atvarious timepoints and at effective doses.

In addition, some mice are treated with antibiotics prior to treatment.For example, vancomycin (0.5 g/L), ampicillin (1.0 g/L), gentamicin (1.0g/L) and amphotericin B (0.2 g/L) are added to the drinking water, andantibiotic treatment is halted at the time of treatment or a few daysprior to treatment. Some immunized mice are treated without receivingantibiotics.

Study animals may be sacrificed by exsanguination from the orbitalplexus under CO₂/O₂ anesthesia, followed by cervical dislocation on day10. For serum preparation, the blood samples are allowed to clot beforecentrifuging. The sera are transferred into clean tubes, each animal ina separate tube. Following exsanguination, of all animals both ears(each ear in a separate vial), the spleen, the mesenteric lymph nodes(MLN), the entire small intestine, and the colon are collected incryovials, snap frozen and stored at <−70° C.

Tissues may be dissociated using dissociation enzymes according to themanufacturer's instructions. Cells are stained for analysis by flowcytometry using techniques known in the art. Staining antibodies caninclude anti-CD11c (dendritic cells), anti-CD80, anti-CD86, anti-CD40,anti-MHCII, anti-CD8a, anti-CD4, and anti-CD103. Other markers that maybe analyzed include pan-immune cell marker CD45, T cell markers (CD3,CD4, CD8, CD25, Foxp3, T-bet, Gata3, Roryt, Granzyme B, CD69, PD-1,CTLA-4), and macrophage/myeloid markers (CD11b, MHCII, CD206, CD40,CSF1R, PD-L1, Gr-1, F4/80). In addition to immunophenotyping, serumcytokines are analyzed including, but not limited to, TNFa, IL-17,IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5, IL-4, IL-2, IL-1b, IFNy,GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES, and MCP-1. Cytokineanalysis may be carried out on immune cells obtained from lymph nodes orother tissue, and/or on purified CD45+ infiltrated immune cells obtainedex vivo. Finally, immunohistochemistry is carried out on various tissuesections to measure T cells, macrophages, dendritic cells, andcheckpoint molecule protein expression.

Example 2: An Evaluation of Test Articles in the Modulation ofDSS-Induced Colitis in C57BL/6 Mice

Dextran sulfate sodium (DSS)-induced colitis is a well-studied animalmodel of colitis, as reviewed by Randhawa et al. (A review onchemical-induced inflammatory bowel disease models in rodents. Korean JPhysiol Pharmacol. 2014. 18(4): 279-288; see also Chassaing et al.Dextran sulfate sodium (DSS)-induced colitis in mice. Curr ProtocImmunol. 2014 Feb. 4; 104: Unit 15.25). In this model, mice are treatedwith DSS in drinking water, resulting in diarrhea and weight loss.

To examine the efficacy of Veillonella in DSS-induced colitis, mice aredivided into groups receiving Veillonella Strain A, Veillonella StrainB, Veillonella Strain C, and/or other Veillonella strain. Groups of miceare treated with DSS to induce colitis as known in the art (Randhawa etal. 2014; Chassaing et al. 2014; see also Kim et al. Investigatingintestinal inflammation in DSS-induced model of IBD. J Vis Exp. 2012.60: 3678). For example, colitis was induced in mice by exposure to 3%DSS-treated drinking water from Day 0 to Day 5. One group does notreceive DSS and serves as naive controls. Animals are dosed with sucrosevehicle (negative control), bacterial strain (1×10⁹ CFU per mouse perday), or anti-p40 positive control (administered i.p. on days 0, 3, 7,and 10). All animals are weighed daily.

In other studies, treatment with a bacterial strain (e.g., a strain ofbacteria listed in Table 1)-containing bacterial composition may beinitiated at some point, either on day 1 of DSS administration, orsometime thereafter. For example, Veillonella may be administered at thesame time as DSS initiation (day 1), or administered upon the firstsigns of disease (e.g. weight loss or diarrhea), or during the stages ofsevere colitis. Mice may be observed daily for weight, morbidity,survival, presence of diarrhea and/or bloody stool.

The bacterial strain is administered at varied dosess, varied intervals,and/or varied routes of administration, and/or in combination with otherVeillonella strains or other species. For example, some mice areintravenously injected with Veillonella at a dose of between 1×10⁴ and5×10⁹ bacterial cells per mouse. While some mice receive the bacteriathrough i.v. injection, other mice may receive bacteria throughintraperitoneal (i.p.) injection, subcutaneous (s.c.) injection, nasalroute administration, oral gavage, or other means of administration.Some mice may receive the bacterial strain every day (e.g. starting onday 1), while others may receive the bacterial strain at alternativeintervals (e.g. every other day, or once every three days). Additionalgroups of mice may receive some ratio of bacterial cells to thebacterial strain. The bacterial cells may be live, dead, or weakened.The bacterial cells may be harvested fresh (or frozen) and administered,or they may be irradiated or heat-killed prior to administration.

The bacterial strain-containing bacterial compositions may be tested fortheir efficacy in a mouse model of DSS-induced colitis, either alone orin combination with whole bacterial cells, with or without the additionof other anti-inflammatory agents.

For example, some groups of mice may receive between 1×10⁴ and 5×10⁹bacterial cells in an administration separate from, or comingled with,the bacterial strain administration. As with the bacterial strain,bacterial cell administration may be varied by route of administration,dose, and schedule. This can include oral gavage, i.v. injection, i.p.injection, or nasal route administration.

Some groups of mice may be treated with additional anti-inflammatoryagent(s) (e.g. anti-CD154, blockade of members of the TNF family, orother treatment), and/or an appropriate control (e.g. vehicle or controlantibody) at various timepoints and at effective doses.

In addition, some mice are treated with antibiotics prior to treatment.For example, vancomycin (0.5 g/L), ampicillin (1.0 g/L), gentamicin (1.0g/L) and amphotericin B (0.2 g/L) are added to the drinking water, andantibiotic treatment is halted at the time of treatment or a few daysprior to treatment. Some mice receive DSS without receiving antibioticsbeforehand.

At various timepoints, mice undergo video endoscopy using a small animalendoscope (Karl Storz Endoskipe, Germany) under isoflurane anesthesia.Still images and video are recorded to evaluate the extent of colitisand the response to treatment. Colitis is scored using criteria known inthe art. Fecal material is collected for study.

The gastrointestinal (GI) tract, lymph nodes, and/or other tissues maybe removed for ex vivo histological, cytokine and/or flow cytometricanalysis using methods known in the art. For example, tissues areharvested and may be dissociated using dissociation enzymes according tothe manufacturer's instructions. Cells are stained for analysis by flowcytometry using techniques known in the art. Staining antibodies caninclude anti-CD11c (dendritic cells), anti-CD80, anti-CD86, anti-CD40,anti-MHCII, anti-CD8a, anti-CD4, and anti-CD103. Other markers that maybe analyzed include pan-immune cell marker CD45, T cell markers (CD3,CD4, CD8, CD25, Foxp3, T-bet, Gata3, Roryt, Granzyme B, CD69, PD-1,CTLA-4), and macrophage/myeloid markers (CD11b, MHCII, CD206, CD40,CSF1R, PD-L1, Gr-1, F4/80). In addition to immunophenotyping, serumcytokines are analyzed including, but not limited to, TNFa, IL-17,IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5, IL-4, IL-2, IL-1b, IFNy,GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES, and MCP-1. Cytokineanalysis may be carried out on immune cells obtained from lymph nodes orother tissue, and/or on purified CD45+GI tract-infiltrated immune cellsobtained ex vivo. Finally, immunohistochemistry is carried out onvarious tissue sections to measure T cells, macrophages, dendriticcells, and checkpoint molecule protein expression.

In order to examine the impact and longevity of disease protection,rather than being sacrificed, some mice may be rechallenged with adisease trigger. Mice are analyzed for susceptibility to colitisseverity following rechallenge.

Following sacrifice, the colon, small intestine, spleen, and mesentericlymph nodes may be collected from all animals, and blood collected foranalysis.

Example 3: A Mouse Model of Experimental Autoimmune Encephalomyelitis(EAE)

EAE is a well-studied animal model of multiple sclerosis, as reviewed byConstantinescu et al. (Experimental autoimmune encephalomyelitis (EAE)as a model for multiple sclerosis (MS). Br J Pharmacol. 2011 October;164(4): 1079-1106). It can be induced in a variety of mouse and ratstrains using different myelin-associated peptides, by the adoptivetransfer of activated encephalitogenic T cells, or the use of TCRtransgenic mice susceptible to EAE, as discussed in Mangalam et al. (Twodiscreet subsets of CD8+ T cells modulate PLP₉₁₋₁₁₀ induced experimentalautoimmune encephalomyelitis in HLA-DR3 transgenic mice. J Autoimmun.2012 June; 38(4): 344-353).

The Veillonella-containing bacterial compositions described herein aretested for their efficacy in the rodent model of EAE, either alone or incombination with whole bacterial cells, with or without the addition ofother anti-inflammatory treatments. For example, female 6-8 week oldC57Bl/6 mice are obtained from Taconic (Germantown, N.Y.). Groups ofmice are administered two subcutaneous (s.c.) injections at two sites onthe back (upper and lower) of 0.1 ml myelin oligodentrocyte glycoprotein35-55 (MOG35-55; 100 ug per injection; 200 ug per mouse (total 0.2 mlper mouse)), emulsified in Complete Freund's Adjuvant (CFA; 2-5 mgkilled Mycobacterium tuberculosis H37Ra/ml emulsion). Approximately 1-2hours after the above, mice are intraperitoneally (i.p.) injected with200 ng Pertussis toxin (PTx) in 0.1 ml PBS (2 ug/ml). An additional IPinjection of PTx is administered on day 2. Alternatively, an appropriateamount of an alternative myelin peptide (e.g. proteolipid protein (PLP))is used to induce EAE. Some animals serve as naïve controls. EAEseverity is assessed and a disability score is assigned daily beginningon day 4 according to methods known in the art (Mangalam et al. 2012).

Treatment with Veillonella Strain A, Veillonella Strain B, VeillonellaStrain C, and/or other Veillonella strain is initiated at some point,either around the time of immunization or following EAE immunization.For example, the bacterial strain-containing bacterial composition maybe administered at the same time as immunization (day 1), or they may beadministered upon the first signs of disability (e.g. limp tail), orduring severe EAE. The bacterial strain-containing bacterialcompositions are administered at varied doses and at defined intervals.For example, some mice are intravenously injected with effective dosesof the bacterial strain. For example, mice may receive between 1×10⁴ and5×10⁹ bacterial cells per mouse. While some mice receive the bacterialstrain through i.v. injection, other mice may receive the bacterialstrain through intraperitoneal (i.p.) injection, subcutaneous (s.c.)injection, nasal route administration, oral gavage, or other means ofadministration. Some mice may receive the bacterial strain every day(e.g. starting on day 1), while others may receive the bacterial strainat alternative intervals (e.g. every other day, or once every threedays). Additional groups of mice may receive some ratio of bacterialcells to the bacterial strain. The bacterial cells may be live, dead, orweakened. The bacterial cells may be harvested fresh (or frozen) andadministered, or they may be irradiated or heat-killed prior toadministration.

For example, some groups of mice may receive between 1×10⁴ and 5×10⁹bacterial cells in an administration separate from, or comingled with,the bacterial strain administration. As with the bacterial strain (e.g.,a strain of bacteria listed in Table 1), bacterial cell administrationmay be varied by route of administration, dose, and schedule. This caninclude oral gavage, i.v. injection, i.p. injection, subcutaneous (s.c.)injection, or nasal route administration.

Some groups of mice may be treated with additional anti-inflammatoryagent(s) or EAE therapeutic(s) (e.g. anti-CD154, blockade of members ofthe TNF family, Vitamin D, or other treatment), and/or an appropriatecontrol (e.g. vehicle or control antibody) at various time points and ateffective doses.

In addition, some mice are treated with antibiotics prior to treatment.For example, vancomycin (0.5 g/L), ampicillin (1.0 g/L), gentamicin (1.0g/L) and amphotericin B (0.2 g/L) are added to the drinking water, andantibiotic treatment is halted at the time of treatment or a few daysprior to treatment. Some immunized mice are treated without receivingantibiotics.

At various time points, mice are sacrificed and sites of inflammation(e.g. brain and spinal cord), lymph nodes, or other tissues may beremoved for ex vivo histological, cytokine and/or flow cytometricanalysis using methods known in the art. For example, tissues aredissociated using dissociation enzymes according to the manufacturer'sinstructions. Cells are stained for analysis by flow cytometry usingtechniques known in the art. Staining antibodies can include anti-CD11c(dendritic cells), anti-CD80, anti-CD86, anti-CD40, anti-MHCII,anti-CD8a, anti-CD4, and anti-CD103. Other markers that may be analyzedinclude pan-immune cell marker CD45, T cell markers (CD3, CD4, CD8,CD25, Foxp3, T-bet, Gata3, Roryt, Granzyme B, CD69, PD-1, CTLA-4), andmacrophage/myeloid markers (CD11b, MHCII, CD206, CD40, CSF1R, PD-L1,Gr-1, F4/80). In addition to immunophenotyping, serum cytokines areanalyzed including, but not limited to, TNFa, IL-17, IL-13, IL-12p70,IL12p40, IL-10, IL-6, IL-5, IL-4, IL-2, IL-1b, IFNy, GM-CSF, G-CSF,M-CSF, MIG, IP10, MIP1b, RANTES, and MCP-1. Cytokine analysis may becarried out on immune cells obtained from lymph nodes or other tissue,and/or on purified CD45+ central nervous system (CNS)-infiltrated immunecells obtained ex vivo. Finally, immunohistochemistry is carried out onvarious tissue sections to measure T cells, macrophages, dendriticcells, and checkpoint molecule protein expression.

In order to examine the impact and longevity of disease protection,rather than being sacrificed, some mice may be rechallenged with adisease trigger (e.g. activated encephalitogenic T cells or re-injectionof EAE-inducing peptides). Mice are analyzed for susceptibility todisease and EAE severity following rechallenge.

Example 4: A Mouse Model of Collagen-Induced Arthritis (CIA)

Collagen-induced arthritis (CIA) is an animal model commonly used tostudy rheumatoid arthritis (RA), as described by Caplazi et al. (Mousemodels of rheumatoid arthritis. Veterinary Pathology. Sep. 1, 2015.52(5): 819-826) (see also Brand et al. Collagen-induced arthritis.Nature Protocols. 2007. 2: 1269-1275; Pietrosimone et al.Collagen-induced arthritis: a model for murine autoimmune arthritis. BioProtoc. 2015 Oct. 20; 5(20): e1626).

Among other versions of the CIA rodent model, one model involvesimmunizing HLA-DQ8 Tg mice with chick type II collagen as described byTaneja et al. (J. Immunology. 2007. 56: 69-78; see also Taneja et al. J.Immunology 2008. 181: 2869-2877; and Taneja et al. Arthritis Rheum.,2007. 56: 69-78). Purification of chick CII has been described by Tanejaet al. (Arthritis Rheum., 2007. 56: 69-78). Mice are monitored for CIAdisease onset and progression following immunization, and severity ofdisease is evaluated and “graded” as described by Wooley, J. Exp. Med.1981. 154: 688-700.

Mice are immunized for CIA induction and separated into varioustreatment groups. The bacterial strain-containing bacterial compositionsare tested for their efficacy in CIA, either alone or in combinationwith whole bacterial cells, with or without the addition of otheranti-inflammatory treatments.

Treatment with the Veillonella-containing bacterial composition isinitiated either around the time of immunization with collagen orpost-immunization. For example, in some groups, the bacterial strain maybe administered at the same time as immunization (day 1), or thebacterial strain may be administered upon first signs of disease, orupon the onset of severe symptoms. The bacterial strain is administeredat varied doses and at defined intervals.

For example, some mice are intravenously injected with Veillonella at adose of between 1×10⁴ and 5×10⁹ bacterial cells per mouse. While somemice receive the bacterial strain through i.v. injection, other groupsof mice may receive the bacterial strain through intraperitoneal (i.p.)injection, subcutaneous (s.c.) injection, nasal route administration,oral gavage, or other means of administration. Some mice may receive thebacterial strain every day (e.g. starting on day 1), while others mayreceive the bacterial strain at alternative intervals (e.g. every otherday, or once every three days). Additional groups of mice may receivesome ratio of bacterial cells to the bacterial strain. The bacterialcells may be live, dead, or weakened. The bacterial cells may beharvested fresh (or frozen) and administered, or they may be irradiatedor heat-killed prior to administration.

For example, some groups of mice may receive between 1×10⁴ and 5×10⁹bacterial cells in an administration separate from, or comingled with,the bacterial strain administration. As with the bacterial strain,bacterial cell administration may be varied by route of administration,dose, and schedule. This can include oral gavage, i.v. injection, i.p.injection, subcutaneous (s.c.) injection, intradermal (i.d.) injection,or nasal route administration.

Some groups of mice may be treated with additional anti-inflammatoryagent(s) or CIA therapeutic(s) (e.g. anti-CD154, blockade of members ofthe TNF family, Vitamin D, or other treatment), and/or an appropriatecontrol (e.g. vehicle or control antibody) at various time points and ateffective doses.

In addition, some mice are treated with antibiotics prior to treatment.For example, vancomycin (0.5 g/L), ampicillin (1.0 g/L), gentamicin (1.0g/L) and amphotericin B (0.2 g/L) are added to the drinking water, andantibiotic treatment is halted at the time of treatment or a few daysprior to treatment. Some immunized mice are treated without receivingantibiotics.

At various time points, serum samples are obtained to assess levels ofanti-chick and anti-mouse CII IgG antibodies using a standard ELISA(Batsalova et al. Comparative analysis of collagen type II-specificimmune responses during development of collagen-induced arthritis in twoB10 mouse strains. Arthritis Res Ther. 2012. 14(6): R237). Also, somemice are sacrificed and sites of inflammation (e.g. synovium), lymphnodes, or other tissues may be removed for ex vivo histological,cytokine and/or flow cytometric analysis using methods known in the art.The synovium and synovial fluid are analyzed for plasma cellinfiltration and the presence of antibodies using techniques known inthe art. In addition, tissues are dissociated using dissociation enzymesaccording to the manufacturer's instructions to examine the profiles ofthe cellular infiltrates. Cells are stained for analysis by flowcytometry using techniques known in the art. Staining antibodies caninclude anti-CD11c (dendritic cells), anti-CD80, anti-CD86, anti-CD40,anti-MHCII, anti-CD8a, anti-CD4, and anti-CD103. Other markers that maybe analyzed include pan-immune cell marker CD45, T cell markers (CD3,CD4, CD8, CD25, Foxp3, T-bet, Gata3, Roryt, Granzyme B, CD69, PD-1,CTLA-4), and macrophage/myeloid markers (CD11b, MHCII, CD206, CD40,CSF1R, PD-L1, Gr-1, F4/80). In addition to immunophenotyping, serumcytokines are analyzed including, but not limited to, TNFa, IL-17,IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5, IL-4, IL-2, IL-1b, IFNy,GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES, and MCP-1. Cytokineanalysis may be carried out on immune cells obtained from lymph nodes orother tissue, and/or on purified CD45+ synovium-infiltrated immune cellsobtained ex vivo. Finally, immunohistochemistry is carried out onvarious tissue sections to measure T cells, macrophages, dendriticcells, and checkpoint molecule protein expression.

In order to examine the impact and longevity of disease protection,rather than being sacrificed, some mice may be rechallenged with adisease trigger (e.g. activated re-injection with CIA-inducingpeptides). Mice are analyzed for susceptibility to disease and CIAseverity following rechallenge.

Example 5: A Mouse Model of Type 1 Diabetes (T1D)

Type 1 diabetes (T1D) is an autoimmune disease in which the immunesystem targets the islets of Langerhans of the pancreas, therebydestroying the body's ability to produce insulin.

There are various models of animal models of T1D, as reviewed by Belleet al. (Mouse models for type 1 diabetes. Drug Discov Today Dis Models.2009; 6(2): 41-45; see also Aileen J F King. The use of animal models indiabetes research. Br J Pharmacol. 2012 June; 166(3): 877-894. There aremodels for chemically-induced T1D, pathogen-induced T1D, as well asmodels in which the mice spontaneously develop T1D.

A Veillonella strain described herein is tested for its efficacy in amouse model of T1D, either alone or in combination with other strains,with or without the addition of other anti-inflammatory treatments.

Depending on the method of T1D induction and/or whether T1D developmentis spontaneous, treatment with the bacterial strain is initiated at somepoint, either around the time of induction or following induction, orprior to the onset (or upon the onset) of spontaneously-occurring T1D.The bacterial strain is administered at varied doses and at definedintervals. For example, some mice are intravenously injected with theVeillonella at a dose of between 1×10⁴ and 5×10⁹ bacterial cells permouse. Other mice may receive 25, 50, or 100 mg of the bacterial strainper mouse. While some mice receive the bacterial strain through i.v.injection, other mice may receive the bacterial strain throughintraperitoneal (i.p.) injection, subcutaneous (s.c.) injection, nasalroute administration, oral gavage, or other means of administration.Some mice may receive the bacterial strain every day, while others mayreceive the bacterial strain at alternative intervals (e.g. every otherday, or once every three days). Additional groups of mice may receivesome ratio of bacterial cells to the bacterial strain. The bacterialcells may be live, dead, or weakened. The bacterial cells may beharvested fresh (or frozen) and administered, or they may be irradiatedor heat-killed prior to administration.

For example, some groups of mice may receive between 1×10⁴ and 5×10⁹bacterial cells in an administration separate from, or comingled with,the bacterial strain administration. As with the bacterial strain,bacterial cell administration may be varied by route of administration,dose, and schedule. This can include oral gavage, i.v. injection, i.p.injection, or nasal route administration.

Some groups of mice may be treated with additional treatments and/or anappropriate control (e.g. vehicle or control antibody) at varioustimepoints and at effective doses.

In addition, some mice are treated with antibiotics prior to treatment.For example, vancomycin (0.5 g/L), ampicillin (1.0 g/L), gentamicin (1.0g/L) and amphotericin B (0.2 g/L) are added to the drinking water, andantibiotic treatment is halted at the time of treatment or a few daysprior to treatment. Some immunized mice are treated without receivingantibiotics.

Blood glucose is monitored biweekly prior to the start of theexperiment. At various timepoints thereafter, nonfasting blood glucoseis measured. At various timepoints, mice are sacrificed and site thepancreas, lymph nodes, or other tissues may be removed for ex vivohistological, cytokine and/or flow cytometric analysis using methodsknown in the art. For example, tissues are dissociated usingdissociation enzymes according to the manufacturer's instructions. Cellsare stained for analysis by flow cytometry using techniques known in theart. Staining antibodies can include anti-CD11c (dendritic cells),anti-CD80, anti-CD86, anti-CD40, anti-MHCII, anti-CD8a, anti-CD4, andanti-CD103. Other markers that may be analyzed include pan-immune cellmarker CD45, T cell markers (CD3, CD4, CD8, CD25, Foxp3, T-bet, Gata3,Roryt, Granzyme B, CD69, PD-1, CTLA-4), and macrophage/myeloid markers(CD11b, MHCII, CD206, CD40, CSF1R, PD-L1, Gr-1, F4/80). In addition toimmunophenotyping, serum cytokines are analyzed including, but notlimited to, TNFa, IL-17, IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5,IL-4, IL-2, IL-1b, IFNy, GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES,and MCP-1. Cytokine analysis may be carried out on immune cells obtainedfrom lymph nodes or other tissue, and/or on purified tissue-infiltratingimmune cells obtained ex vivo. Finally, immunohistochemistry is carriedout on various tissue sections to measure T cells, macrophages,dendritic cells, and checkpoint molecule protein expression. Antibodyproduction may also be assessed by ELISA.

In order to examine the impact and longevity of disease protection,rather than being sacrificed, some mice may be rechallenged with adisease trigger, or assessed for susceptibility to relapse. Mice areanalyzed for susceptibility to diabetes onset and severity followingrechallenge (or spontaneously-occurring relapse).

Example 6: A Mouse Model of Primary Sclerosing Cholangitis (PSC)

Primary Sclerosing Cholangitis (PSC) is a chronic liver disease thatslowly damages the bile ducts and leads to end-stage cirrhosis. It isassociated with inflammatory bowel disease (IBD).

There are various animal models for PSC, as reviewed by Fickert et al.(Characterization of animal models for primary sclerosing cholangitis(PSC). J Hepatol. 2014 June. 60(6): 1290-1303; see also Pollheimer andFickert. Animal models in primary biliary cirrhosis and primarysclerosing cholangitis. Clin Rev Allergy Immunol. 2015 June 48(2-3):207-17). Induction of disease in PSC models includes chemical induction(e.g. 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-inducedcholangitis), pathogen-induced (e.g. Cryptosporidium parvum),experimental biliary obstruction (e.g. common bile duct ligation(CBDL)), and transgenic mouse model of antigen-driven biliary injury(e.g. Ova-Bil transgenic mice). For example, bile duct ligation isperformed as described by Georgiev et al. (Characterization oftime-related changes after experimental bile duct ligation. Br J Surg.2008. 95(5): 646-56), or disease is induced by DCC exposure as describedby Fickert et al. (A new xenobiotic-induced mouse model of sclerosingcholangitis and biliary fibrosis. Am J Path. Vol 171(2): 525-536.

A Veillonella strain described herein is tested for its efficacy in amouse model of PSC, either alone or in combination with other strains,with or without the addition of some other therapeutic agent.

DCC-Induced Cholangitis

For example, 6-8 week old C57bl/6 mice are obtained from Taconic orother vendor. Mice are fed a 0.1% DCC-supplemented diet for variousdurations. Some groups receive DCC-supplement food for 1 week, othersfor 4 weeks, others for 8 weeks. Some groups of mice may receive aDCC-supplemented diet for a length of time and then be allowed torecover, thereafter receiving a normal diet. These mice may be studiedfor their ability to recover from disease and/or their susceptibility torelapse upon subsequent exposure to DCC. Treatment with VeillonellaStrain A, Veillonella Strain B, Veillonella Strain C, and/or otherVeillonella Strain is initiated at some point, either around the time ofDCC-feeding or subsequent to initial exposure to DCC. For example, thebacterial strain may be administered on day 1, or they may beadministered sometime thereafter. The bacterial strain is administeredat varied doses and at defined intervals. For example, some mice areintravenously injected with the bacterial strain at a range between1×10⁴ and 5×10⁹ bacterial cells per mouse. Other mice may receive 25,50, 100 mg of the bacterial strain per mouse. While some mice receivethe bacterial strain through i.v. injection, other mice may receive thebacterial strain through i.p. injection, subcutaneous (s.c.) injection,nasal route administration, oral gavage, or other means ofadministration. Some mice may receive the bacterial strain every day(e.g. starting on day 1), while others may receive the bacterial strainat alternative intervals (e.g. every other day, or once every threedays). Additional groups of mice may receive some ratio of bacterialcells to the bacterial strain. The bacterial cells may be live, dead, orweakened. The bacterial cells may be harvested fresh (or frozen), andadministered, or they may be irradiated or heat-killed prior toadministration. For example, some groups of mice may receive between1×10⁴ and 5×10⁹ bacterial cells in an administration separate from, orcomingled with, the bacterial strain administration. Veillonellaadministration may be varied by route of administration, dose, andschedule. This can include oral gavage, i.v. injection, i.p. injection,or nasal route administration. Some groups of mice may be treated withadditional agents and/or an appropriate control (e.g. vehicle orantibody) at various timepoints and at effective doses.

In addition, some mice are treated with antibiotics prior to treatment.For example, vancomycin (0.5 g/L), ampicillin (1.0 g/L), gentamicin (1.0g/L) and amphotericin B (0.2 g/L) are added to the drinking water, andantibiotic treatment is halted at the time of treatment or a few daysprior to treatment. Some immunized mice are treated without receivingantibiotics. At various timepoints, serum samples are analyzed for ALT,AP, bilirubin, and serum bile acid (BA) levels.

At various timepoints, mice are sacrificed, body and liver weight arerecorded, and sites of inflammation (e.g. liver, small and largeintestine, spleen), lymph nodes, or other tissues may be removed for exvivo histolomorphological characterization, cytokine and/or flowcytometric analysis using methods known in the art (see Fickert et al.Characterization of animal models for primary sclerosing cholangitis(PSC)). J Hepatol. 2014. 60(6): 1290-1303). For example, bile ducts arestained for expression of ICAM-1, VCAM-1, MadCAM-1. Some tissues arestained for histological examination, while others are dissociated usingdissociation enzymes according to the manufacturer's instructions. Cellsare stained for analysis by flow cytometry using techniques known in theart. Staining antibodies can include anti-CD11c (dendritic cells),anti-CD80, anti-CD86, anti-CD40, anti-MHCII, anti-CD8a, anti-CD4, andanti-CD103. Other markers that may be analyzed include pan-immune cellmarker CD45, T cell markers (CD3, CD4, CD8, CD25, Foxp3, T-bet, Gata3,Roryt, Granzyme B, CD69, PD-1, CTLA-4), and macrophage/myeloid markers(CD11b, MHCII, CD206, CD40, CSF1R, PD-L1, Gr-1, F4/80), as well asadhesion molecule expression (ICAM-1, VCAM-1, MadCAM-1). In addition toimmunophenotyping, serum cytokines are analyzed including, but notlimited to, TNFa, IL-17, IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5,IL-4, IL-2, IL-1b, IFNy, GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES,and MCP-1. Cytokine analysis may be carried out on immune cells obtainedfrom lymph nodes or other tissue, and/or on purified CD45+ bileduct-infiltrated immune cells obtained ex vivo.

Liver tissue is prepared for histological analysis, for example, usingSirius-red staining followed by quantification of the fibrotic area. Atthe end of the treatment, blood is collected for plasma analysis ofliver enzymes, for example, AST or ALT, and to determine Bilirubinlevels. The hepatic content of Hydroxyproline can be measured usingestablished protocols. Hepatic gene expression analysis of inflammationand fibrosis markers may be performed by qRT-PCR using validatedprimers. These markers may include, but are not limited to, MCP-1,alpha-SMA, Coll1a1, and TIMP-. Metabolite measurements may be performedin plasma, tissue and fecal samples using established metabolomicsmethods. Finally, immunohistochemistry is carried out on liver sectionsto measure neutrophils, T cells, macrophages, dendritic cells, or otherimmune cell infiltrates.

In order to examine the impact and longevity of disease protection,rather than being sacrificed, some mice may be rechallenged with DCC ata later time. Mice are analyzed for susceptibility to cholangitis andcholangitis severity following rechallenge.

BDL-Induced Cholangitis

Alternatively, Veillonella-containing bacterial compositions are testedfor their efficacy in BDL-induced cholangitis. For example, 6-8 week oldC57Bl/6J mice are obtained from Taconic or other vendor. After anacclimation period the mice are subjected to a surgical procedure toperform a bile duct ligation (BDL). Some control animals receive a shamsurgery. The BDL procedure leads to liver injury, inflammation andfibrosis within 7-21 days.

Treatment with Veillonella is initiated at some point, either around thetime of surgery or some time following the surgery. Veillonella isadministered at varied doses and at defined intervals. For example, somemice are intravenously injected with the bacterial strain at a rangebetween 1×10⁴ and 5×10⁹ bacterial cells per mouse. Other mice mayreceive 25, 50, or 100 mg of the bacterial strain per mouse. While somemice receive Veillonella through i.v. injection, other mice may receivethe bacterial strain through i.p. injection, subcutaneous (s.c.)injection, nasal route administration, oral gavage, or other means ofadministration. Some mice receive the bacterial strain every day (e.g.starting on day 1), while others may receive the bacterial strain atalternative intervals (e.g. every other day, or once every three days).Additional groups of mice may receive some ratio of bacterial cells tothe bacterial strain. The bacterial cells may be live, dead, orweakened. They bacterial cells may be harvested fresh (or frozen), andadministered, or they may be irradiated or heat-killed prior toadministration. For example, some groups of mice may receive between1×10⁴ and 5×10⁹ bacterial cells in an administration separate from, orcomingled with, the bacterial strain administration. As with thebacterial strain, bacterial cell administration may be varied by routeof administration, dose, and schedule. This can include oral gavage,i.v. injection, i.p. injection, or nasal route administration. Somegroups of mice may be treated with additional agents and/or anappropriate control (e.g. vehicle or antibody) at various timepoints andat effective doses.

In addition, some mice are treated with antibiotics prior to treatment.For example, vancomycin (0.5 g/L), ampicillin (1.0 g/L), gentamicin (1.0g/L) and amphotericin B (0.2 g/L) are added to the drinking water, andantibiotic treatment is halted at the time of treatment or a few daysprior to treatment. Some immunized mice are treated without receivingantibiotics. At various timepoints, serum samples are analyzed for ALT,AP, bilirubin, and serum bile acid (BA) levels.

At various timepoints, mice are sacrificed, body and liver weight arerecorded, and sites of inflammation (e.g. liver, small and largeintestine, spleen), lymph nodes, or other tissues may be removed for exvivo histolomorphological characterization, cytokine and/or flowcytometric analysis using methods known in the art (see Fickert et al.Characterization of animal models for primary sclerosing cholangitis(PSC)). J Hepatol. 2014. 60(6): 1290-1303). For example, bile ducts arestained for expression of ICAM-1, VCAM-1, MadCAM-1. Some tissues arestained for histological examination, while others are dissociated usingdissociation enzymes according to the manufacturer's instructions. Cellsare stained for analysis by flow cytometry using techniques known in theart. Staining antibodies can include anti-CD11c (dendritic cells),anti-CD80, anti-CD86, anti-CD40, anti-MHCII, anti-CD8a, anti-CD4, andanti-CD103. Other markers that may be analyzed include pan-immune cellmarker CD45, T cell markers (CD3, CD4, CD8, CD25, Foxp3, T-bet, Gata3,Roryt, Granzyme B, CD69, PD-1, CTLA-4), and macrophage/myeloid markers(CD11b, MHCII, CD206, CD40, CSF1R, PD-L1, Gr-1, F4/80), as well asadhesion molecule expression (ICAM-1, VCAM-1, MadCAM-1). In addition toimmunophenotyping, serum cytokines are analyzed including, but notlimited to, TNFa, IL-17, IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5,IL-4, IL-2, IL-1b, IFNy, GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES,and MCP-1. Cytokine analysis may be carried out on immune cells obtainedfrom lymph nodes or other tissue, and/or on purified CD45+ bileduct-infiltrated immune cells obtained ex vivo.

Liver tissue is prepared for histological analysis, for example, usingSirius-red staining followed by quantification of the fibrotic area. Atthe end of the treatment, blood is collected for plasma analysis ofliver enzymes, for example, AST or ALT, and to determine Bilirubinlevels. The hepatic content of Hydroxyproline can be measured usingestablished protocols. Hepatic gene expression analysis of inflammationand fibrosis markers may be performed by qRT-PCR using validatedprimers. These markers may include, but are not limited to, MCP-1,alpha-SMA, Coll1a1, and TIMP-. Metabolite measurements may be performedin plasma, tissue and fecal samples using established metabolomicsmethods. Finally, immunohistochemistry is carried out on liver sectionsto measure neutrophils, T cells, macrophages, dendritic cells, or otherimmune cell infiltrates.

In order to examine the impact and longevity of disease protection,rather than being sacrificed, some mice may be analyzed for recovery.

Example 7: A Mouse Model of Nonalcoholic Steatohepatitis (NASH)

Nonalcoholic Steatohepatitis (NASH) is a severe form of NonalcoholicFatty Liver Disease (NAFLD), where buildup of hepatic fat (steatosis)and inflammation lead to liver injury and hepatocyte cell death(ballooning).

There are various animal models of NASH, as reviewed by Ibrahim et al.(Animal models of nonalcoholic steatohepatitis: Eat, Delete, andInflame. Dig Dis Sci. 2016 May. 61(5): 1325-1336; see also Lau et al.Animal models of non-alcoholic fatty liver disease: current perspectivesand recent advances 2017 January 241(1): 36-44).

Veillonella is tested for its efficacy in a mouse model of NASH, eitheralone or in combination with whole bacterial cells, with or without theaddition of another therapeutic agent. For example, 8-10 week oldC57Bl/6J mice, obtained from Taconic (Germantown, N.Y.), or othervendor, are placed on a methionine choline deficient (MCD) diet for aperiod of 4-8 weeks during which NASH features develop, includingsteatosis, inflammation, ballooning and fibrosis.

Treatment with Veillonella Strain A, Veillonella Strain B, VeillonellaStrain C, and/or other Veillonella strain is initiated at some point,either at the beginning of the diet, or at some point following dietinitiation (for example, one week after). For example, the bacterialstrain may be administered starting in the same day as the initiation ofthe MCD diet. The bacterial strain is administered at varied doses andat defined intervals. For example, some mice are intravenously injectedwith the bacterial strain at doses between 1×10⁴ and 5×10⁹ bacterialcells per mouse. Other mice may receive 25, 50, or 100 mg of thebacterial strain per mouse. While some mice receive the bacterial strainthrough i.v. injection, other mice may receive the bacterial strainthrough intraperitoneal (i.p.) injection, subcutaneous (s.c.) injection,nasal route administration, oral gavage, or other means ofadministration. Some mice may receive the bacterial strain every day(e.g. starting on day 1), while others may receive the bacterial strainat alternative intervals (e.g. every other day, or once every threedays). Additional groups of mice may receive some ratio of bacterialcells to the bacterial strain. The bacterial cells may be live, dead, orweakened. The bacterial cells may be harvested fresh (or frozen) andadministered, or they may be irradiated or heat-killed prior toadministration.

For example, some groups of mice may receive between 1×10⁴ and 5×10⁹bacterial cells in an administration separate from, or comingled with,the bacterial strain administration. As with the bacterial strain,bacterial cell administration may be varied by route of administration,dose, and schedule. This can include oral gavage, i.v. injection, i.p.injection, or nasal route administration. Some groups of mice may betreated with additional NASH therapeutic(s) (e.g., FXR agonists, PPARagonists, CCR2/5 antagonists or other treatment) and/or appropriatecontrol at various timepoints and effective doses.

At various timepoints and/or at the end of the treatment, mice aresacrificed and liver, intestine, blood, feces, or other tissues may beremoved for ex vivo histological, biochemical, molecular or cytokineand/or flow cytometry analysis using methods known in the art. Forexample, liver tissues are weighed and prepared for histologicalanalysis, which may comprise staining with H&E, Sirius Red, anddetermination of NASH activity score (NAS). At various timepoints, bloodis collected for plasma analysis of liver enzymes, for example, AST orALT, using standards assays. In addition, the hepatic content ofcholesterol, triglycerides, or fatty acid acids can be measured usingestablished protocols. Hepatic gene expression analysis of inflammation,fibrosis, steatosis, ER stress, or oxidative stress markers may beperformed by qRT-PCR using validated primers. These markers may include,but are not limited to, IL-6, MCP-1, alpha-SMA, Coll1a1, CHOP, and NRF2.Metabolite measurements may be performed in plasma, tissue and fecalsamples using established biochemical and mass-spectrometry-basedmetabolomics methods. Serum cytokines are analyzed including, but notlimited to, TNFa, IL-17, IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5,IL-4, IL-2, IL-1b, IFNy, GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES,and MCP-1. Cytokine analysis may be carried out on immune cells obtainedfrom lymph nodes or other tissue, and/or on purified CD45+ bileduct-infiltrated immune cells obtained ex vivo. Finally,immunohistochemistry is carried out on liver or intestine sections tomeasure neutrophils, T cells, macrophages, dendritic cells, or otherimmune cell infiltrates.

In order to examine the impact and longevity of disease protection,rather than being sacrificed, some mice may be analyzed for recovery.

Example 8: A Mouse Model of Psoriasis

Psoriasis is a T-cell-mediated chronic inflammatory skin disease.So-called “plaque-type” psoriasis is the most common form of psoriasisand is typified by dry scales, red plaques, and thickening of the skindue to infiltration of immune cells into the dermis and epidermis.Several animal models have contributed to the understanding of thisdisease, as reviewed by Gudjonsson et al. (Mouse models of psoriasis. JInvest Derm. 2007. 127: 1292-1308; see also van der Fits et al.Imiquimod-induced psoriasis-like skin inflammation in mice is mediatedvia the IL-23/IL-17 axis. J. Immunol. 2009 May 1. 182(9): 5836-45).

Psoriasis can be induced in a variety of mouse models, including thosethat use transgenic, knockout, or xenograft models, as well as topicalapplication of imiquimod (IMQ), a TLR7/8 ligand.

Veillonella is tested for its efficacy in the mouse model of psoriasis,either alone or in combination with whole bacterial cells, with orwithout the addition of other anti-inflammatory treatments. For example,6-8 week old C57Bl/6 or Balb/c mice are obtained from Taconic(Germantown, N.Y.), or other vendor. Mice are shaved on the back and theright ear. Groups of mice receive a daily topical dose of 62.5 mg ofcommercially available IMQ cream (5%) (Aldara; 3M Pharmaceuticals). Thedose is applied to the shaved areas for 5 or 6 consecutive days. Atregular intervals, mice are scored for erythema, scaling, and thickeningon a scale from 0 to 4, as described by van der Fits et al. (2009). Miceare monitored for ear thickness using a Mitutoyo micrometer.

Treatment with the bacterial strain is initiated at some point, eitheraround the time of the first application of IMQ, or somethingthereafter. For example, Veillonella may be administered at the sametime as the subcutaneous injections (day 0), or they may be administeredprior to, or upon, application. The bacterial strain is administered atvaried doses and at defined intervals. For example, some mice areintravenously injected with the bacterial strain at a dose of between1×10⁴ and 5×10⁹ bacterial cells per mouse. Other mice may receive 25,50, or 100 mg of the bacterial strain per mouse. While some mice receivethe bacterial strain through i.v. injection, other mice may receive thebacterial strain through intraperitoneal (i.p.) injection, nasal routeadministration, oral gavage, topical administration, intradermal (i.d.)injection, subcutaneous (s.c.) injection, or other means ofadministration. Some mice may receive the bacterial strain every day(e.g. starting on day 0), while others may receive the bacterial strainat alternative intervals (e.g. every other day, or once every threedays). Additional groups of mice may receive some ratio of bacterialcells to the bacterial strain. The bacterial cells may be live, dead, orweakened. The bacterial cells may be harvested fresh (or frozen) andadministered, or they may be irradiated or heat-killed prior toadministration.

For example, some groups of mice may receive between 1×10⁴ and 5×10⁹bacterial cells in an administration separate from, or comingled with,the bacterial strain administration. As with the bacterial strain,bacterial cell administration may be varied by route of administration,dose, and schedule. This can include oral gavage, i.v. injection, i.p.injection, i.d. injection, s.c. injection, topical administration, ornasal route administration.

Some groups of mice may be treated with anti-inflammatory agent(s) (e.g.anti-CD154, blockade of members of the TNF family, or other treatment),and/or an appropriate control (e.g. vehicle or control antibody) atvarious timepoints and at effective doses.

In addition, some mice are treated with antibiotics prior to treatment.For example, vancomycin (0.5 g/L), ampicillin (1.0 g/L), gentamicin (1.0g/L) and amphotericin B (0.2 g/L) are added to the drinking water, andantibiotic treatment is halted at the time of treatment or a few daysprior to treatment. Some immunized mice are treated without receivingantibiotics.

At various timepoints, samples from back and ear skin are taken forcryosection staining analysis using methods known in the art. Othergroups of mice are sacrificed and lymph nodes, spleen, mesenteric lymphnodes (MLN), the small intestine, colon, and other tissues may beremoved for histology studies, ex vivo histological, cytokine and/orflow cytometric analysis using methods known in the art. Some tissuesmay be dissociated using dissociation enzymes according to themanufacturer's instructions. Cryosection samples, tissue samples, orcells obtained ex vivo are stained for analysis by flow cytometry usingtechniques known in the art. Staining antibodies can include anti-CD11c(dendritic cells), anti-CD80, anti-CD86, anti-CD40, anti-MHCII,anti-CD8a, anti-CD4, and anti-CD103. Other markers that may be analyzedinclude pan-immune cell marker CD45, T cell markers (CD3, CD4, CD8,CD25, Foxp3, T-bet, Gata3, Roryt, Granzyme B, CD69, PD-1, CTLA-4), andmacrophage/myeloid markers (CD11b, MHCII, CD206, CD40, CSF1R, PD-L1,Gr-1, F4/80). In addition to immunophenotyping, serum cytokines areanalyzed including, but not limited to, TNFa, IL-17, IL-13, IL-12p70,IL12p40, IL-10, IL-6, IL-5, IL-4, IL-2, IL-1b, IFNy, GM-CSF, G-CSF,M-CSF, MIG, IP10, MIP1b, RANTES, and MCP-1. Cytokine analysis may becarried out on immune cells obtained from lymph nodes or other tissue,and/or on purified CD45+ skin-infiltrated immune cells obtained ex vivo.Finally, immunohistochemistry is carried out on various tissue sectionsto measure T cells, macrophages, dendritic cells, and checkpointmolecule protein expression.

In order to examine the impact and longevity of psoriasis protection,rather than being sacrificed, some mice may be studied to assessrecovery, or they may be rechallenged with IMQ. The groups ofrechallenged mice are analyzed for susceptibility to psoriasis andseverity of response.

Example 9: A Mouse Melanoma Model

Female 6-8 week old C57Bl/6 mice are obtained from Taconic (Germantown,N.Y.). 100,000 B16-F10 (ATCC CRL-6475) tumor cells are resuspended insterile PBS containing 50% Matrigel and inoculated in a 100 ul finalvolume into one hind flank (the first flank) of each mouse. Treatmentwith Veillonella Strain A, Veillonella Strain B, Veillonella Strain C,and/or other Veillonella strain is initiated at some point followingtumor cell inoculation at varied doses and at defined intervals. Forexample, some mice receive between 1-5×10{circumflex over ( )}9 CFU (100μl final volume) per dose. Possible routes of administration includeoral gavage (p.o.), intravenous injection, intratumoral injection (IT)or peritumoral or subtumoral or subcutaneous injection. In order toassess the systemic anti-tumoral effects of Veillonella treatment,additional mice may be inoculated with tumor cells in the contralateral(untreated, second) flank prior to IT, peritumoral, or subtumoraltreatment with Veillonella in the first flank.

Some mice may receive Veillonella (p.o.) on day 1 (the day followingtumor cell injection). Other mice may receive seven (7) consecutivedoses of a bacterial strain (one dose per day on days 14-21). Other micereceive daily dosing or, alternatively, some mice receive dosing everyother day. Alternatively, mice are randomized into various treatmentgroups at a defined timepoint (e.g. on day 13) or when the tumors reacha certain size (e.g. 100 mm³) and treatment is then initiatedaccordingly. For example, when tumor volumes reach an average of 100 mm³(approximately 10-12 days following tumor cell inoculation), animals aredistributed into groups and treated with either vehicle or a bacterialstrain (p.o. or IT). Some additional groups of mice may be treated withan additional cancer therapeutic or appropriate control antibody. Oneexample of a cancer therapeutic that may be administered is an inhibitorof an immune checkpoint, for example anti-PD-1, anti-PD-L1, or othertreatment that blocks the binding of an immune checkpoint to itsligand(s). Checkpoint inhibitors anti-PD-1 and anti-PD-L1 may beformulated in PBS and administered intraperitoneally (i.p.) in effectivedoses. For example, mice are given 100 ug of anti-PD-1 (i.p.) every fourdays starting on day 1, and continuing for the duration of the study.

In addition, some mice are treated with antibiotics prior to treatment.For example, vancomycin (0.5 g/L), ampicillin (1.0 g/L), gentamicin (1.0g/L) and amphotericin B (0.2 g/L) are added to the drinking water, andantibiotic treatment is halted at the time of treatment or a few daysprior to treatment. Some mice are inoculated with tumor cells withoutreceiving prior treatment with antibiotics.

At various timepoints, mice are sacrificed and tumors, lymph nodes, orother tissues may be removed for ex vivo flow cytometric analysis usingmethods known in the art. For example, tumors are dissociated using aMiltenyi tumor dissociation enzyme cocktail according to themanufacturer's instructions. Tumor weights are recorded and tumors arechopped then placed in 15 ml tubes containing the enzyme cocktail andplaced on ice. Samples are then placed on a gentle shaker at 37° C. for45 minutes and quenched with up to 15 ml complete RPMI. Each cellsuspension is strained through a 70 μm filter into a 50 ml falcon tubeand centrifuged at 1000 rpm for 10 minutes. Cells are resuspended inFACS buffer and washed to remove remaining debris. If necessary, samplesare strained again through a second 70 μm filter into a new tube. Cellsare stained for analysis by flow cytometry using techniques known in theart. Staining antibodies can include anti-CD11c (dendritic cells),anti-CD80, anti-CD86, anti-CD40, anti-MHCII, anti-CD8a, anti-CD4, andanti-CD103. Other markers that may be analyzed include pan-immune cellmarker CD45, T cell markers (CD3, CD4, CD8, CD25, Foxp3, T-bet, Gata3,Roryt, Granzyme B, CD69, PD-1, CTLA-4), and macrophage/myeloid markers(CD11b, MHCII, CD206, CD40, CSF1R, PD-L1, Gr-1). In addition toimmunophenotyping, serum cytokines are analyzed including, but notlimited to, TNFa, IL-17, IL-13, IL-12p70, IL12p40, IL-10, IL-6, IL-5,IL-4, IL-2, IL-1b, IFNy, GM-CSF, G-CSF, M-CSF, MIG, IP10, MIP1b, RANTES,and MCP-1. Cytokine analysis may be carried out immune cells obtainedfrom lymph nodes or other tissue, and/or on purified CD45+tumor-infiltrated immune cells obtained ex vivo. Finally,immunohistochemistry is carried out on tumor sections to measure Tcells, macrophages, dendritic cells, and checkpoint molecule proteinexpression.

Rather than being sacrificed, some mice may be rechallenged with tumorcell injection into the contralateral flank (or other area) to determinethe impact of the immune system's memory response on tumor growth.

In mice receiving the MCD (NASH-inducing) diet, orally administeredVeillonella Strain C was efficacious in reducing the NAS score comparedto vehicle and no treatment groups (negative controls) (FIG. 10).Veillonella Strain C reduced the fibrosis score in treated mice (FIG.11). Veillonella Strain C reduced hepatic total cholesterol (FIG. 12)and hepatic Triglycerides (FIG. 13).

Example 10: A Mouse Lung Cancer Model

Veillonella is tested for its efficacy in the mouse lung cancer model,either alone or in combination with other cancer therapies, includingcheckpoint inhibitor(s). Mice are divided into groups receivingVeillonella Strain A, Veillonella Strian B, Veillonella Strain C, and/orother Veillonella strain, with or without checkpoint inhibitortreatment. As described in Example 9, Veillonella is administered atvaried doses at defined intervals. For example, some mice receive abacterial strain (p.o.) on the day following tumor cell injection (day1). Some mice receive seven (7) consecutive doses of a bacterial strain(one dose per day on days 14-21). Other mice receive daily dosing or,alternatively, some mice receive dosing every other day. Alternatively,mice are randomized into various treatment groups at a defined timepoint(e.g. on day 13) or when the tumors reach a certain size (e.g. 100 mm³)and treatment is then initiated accordingly.

1×10⁶ LLC1 cells, or an appropriate number of lung cancer cells fromanother lung cancer cell line, are injected into the hind flank ofsyngeneic mice. Tumors from the various treatment groups are measuredwith calipers at regular intervals. As described in Example 9, some miceare sacrificed for ex vivo tumor analysis using flow cytometry. Othermice may be rechallenged with tumor cell injection into thecontralateral flank to determine the impact of the immune system'smemory response on tumor growth.

Example 11: A Mouse Breast Cancer Model

A Veillonella strain is tested for its efficacy in the mouse breastcancer model, either alone or in combination with other cancertherapies, including checkpoint inhibitor(s). Mice are divided intogroups receiving Veillonella Strain A, Veillonella Strain B, VeillonellaStrain C, and/or other Veillonella strain, with or without checkpointinhibitor treatment. As described in Example 9, a bacterial strain isadministered at varied doses at defined intervals. For example, somemice receive a bacterial strain (p.o.) on the day following tumor cellinjection (day 1). Some mice receive seven (7) consecutive doses of abacterial strain (one dose per day on days 14-21). Other mice receivedaily dosing or, alternatively, some mice receive dosing every otherday. Alternatively, mice are randomized into various treatment groups ata defined timepoint (e.g. on day 13) or when the tumors reach a certainsize (e.g. 100 mm³) and treatment is then initiated accordingly.

4T1 mouse mammary carcinoma cells are obtained from ATCC and 1×10⁶ cellsin 50 ul PBS are injected subcutaneously into one or both hind limbs ofBalb/c female mice (as described by Wang et al. 2003, Systemicdissemination of viral vectors during intratumoral injection. MolecularCancer Therapeutics; 2(11)). Alternatively, EMT6 mouse mammary carcinomacells are obtained from ATCC and 1×10⁶ cells in 50 μl PBS are injectedsubcutaneously into one or both of the hind limbs of Balb/c female mice6-8 weeks old (as described by Guo et al. 2014, CombinatorialPhotothermal and Immuno Cancer Therapy Using Chitosan-Coated HollowCopper Sulfide Nanoparticles. ASC Nano.; 8(6): 5670-5681). In addition,other available mouse mammary cell lines may be used.

Tumors from the various treatment groups are measured with calipers atregular intervals. As described in Example 9, Veillonella isadministered at varied doses at defined intervals. For example, somemice are sacrificed for ex vivo tumor analysis using flow cytometry.Other mice may be rechallenged with tumor cell injection into thecontralateral flank to determine the impact of the immune system'smemory response on tumor growth.

Alternatively, 4T1 cells can be used in an orthotopic murine model ofbreast cancer as described by Tao et al. (Tao et al. 2008. Imagable 4T1model for the study of late stage breast cancer. 8: 288). Mice aresacrificed for ex vivo tumor analysis. Tumors are analyzed by flowcytometry and immunohistochemistry.

Example 12: A Mouse Pancreatic Cancer Model

A Veillonella strain is tested for its efficacy in the mouse model ofpancreatic cancer, either alone or in combination with other cancertherapies, including checkpoint inhibitor(s). Mice are divided intogroups receiving a bacterial strain, with or without checkpointinhibitor treatment. As described in Example 9, some mice receiveVeillonella (p.o.) on the day following tumor cell injection (day 1).Some mice receive seven (7) consecutive doses of a bacterial strain (onedose per day on days 14-21). Other mice receive daily dosing or,alternatively, some mice receive dosing every other day. Alternatively,mice are randomized into various treatment groups at a defined timepoint(e.g. on day 13) or when the tumors reach a certain size (e.g. 100 mm³)and treatment is then initiated accordingly.

Panc02 cells are maintained in DMEM, supplemented with 10% fetal calfserum and 1% penicillin/streptomycin, and incubated at 37° C. at 5% CO2.Female 8-10 week-old C57Bl/6 mice are obtained from Charles River, Inc.or other certified vendor. Female C57Bl/6 mice are injectedsubcutaneously into the right hind flank with 1×10⁶ Panc02 cells. Thisprotocol is based on standard Panc02 tumor models (Maletzki et al. 2008.Pancreatic cancer regression by intratumoral injection of liveStreptococcus pyogenes in a syngeneic mouse model. Gut. 57:483-491).Tumors from the various treatment groups are measured with calipers atregular intervals. As described in Example 9, some mice are sacrificedfor ex vivo tumor analysis using flow cytometry, while other mice arerechallenged to determine the impact of the memory response on tumorgrowth.

Alternatively, Panc02, 6606PDA, or Capan-1 cells lines can be used in anorthotopic murine model of pancreatic cancer as described by Partecke etal. (Partecke et al. 2011. A syngeneic orthotopic murine model ofpancreatic adenocarcinoma in the C57/Bl6 mouse using the Panc02 and6606PDA cell lines. Eur. Surg. Res. 47(2):98-107) or Chai et al. (Chaiet al. 2013. Bioluminescent orthotopic model of pancreatic cancerprogression. J. Vis. Exp. 76: 50395). Mice are sacrificed for ex vivotumor analysis. Tumors are analyzed by flow cytometry andimmunohistochemistry.

Example 13: A Mouse Model of Hepatocellular Carcinoma

A Veillonella strain is tested for its efficacy in the mouse model ofhepatocellular carcinoma, either alone or in combination with othercancer therapies, including checkpoint inhibitor(s). Mice are dividedinto groups receiving a bacterial strain, with or without checkpointinhibitor treatment. As described in Example 9, Veillonella isadministered at varied doses at defined intervals. For example, somemice receive Veillonella (p.o.) on the day following tumor cellinjection (day 1). Some mice receive seven (7) consecutive doses of abacterial strain (one dose per day on days 14-21). Other mice receivedaily dosing or, alternatively, some mice receive dosing every otherday. Alternatively, mice are randomized into various treatment groups ata defined timepoint (e.g. on day 13) or when the tumors reach a certainsize (e.g. 100 mm³) and treatment is then initiated accordingly.

Hepatocellular carcinoma is induced in mice by subcutaneous inoculationof 1×10⁶ Hepa129 cells (obtained from NCI or other source), or anappropriate number of cells from other hepatocellular carcinoma cellline (as described by Gonzalez-Carmona et al. 2008. CD40ligand-expressing dendritic cells induce regression of hepatocellularcarcinoma by activating innate and acquired immunity in vivo.Hepatology. 48(1):157-168). Tumor cells are inoculated into one or bothflanks. Tumors from the various treatment groups are measured withcalipers at regular intervals. As described in Example 9, some mice aresacrificed for ex vivo tumor analysis using flow cytometry, while othermice are rechallenged to determine the impact of the memory response ontumor growth.

Example 14: A Mouse Lymphoma Model

A Veillonella strain is tested for its efficacy in the mouse model oflymphoma, either alone or in combination with other cancer therapies,including checkpoint inhibitor(s). For example, mice are divided intogroups receiving Veillonella Strain A, Veillonella Strain B, VeillonellaStrain C, and/or other Veillonella strain, with or without checkpointinhibitor treatment. As described in Example 9, A bacterial strain isadministered at varied doses at defined intervals. For example, somemice receive A bacterial strain (p.o.) on the day following tumor cellinjection (day 1). Some mice receive seven (7) consecutive doses of Abacterial strain (one dose per day on days 14-21). Other mice receivedaily dosing or, alternatively, some mice receive dosing every otherday. Alternatively, mice are randomized into various treatment groups ata defined timepoint (e.g. on day 13) or when the tumors reach a certainsize (e.g. 100 mm³) and treatment is then initiated accordingly.

One lymphoma cell line is the A20 lymphoma, although other lymphoma celllines may be used with syngeneic mice. A20 lymphoma cells are obtainedfrom ATCC and 5×10⁶ cells in 50 ul PBS are injected subcutaneously intoone or both of the hind limbs of Balb/c female mice (as described byHouot et al. 2009. T-cell modulation combined with intratumoral CpGcures lymphoma in a mouse model without the need for chemotherapy.Blood. 113(15): 3546-3552). Tumors from the various treatment groups aremeasured with calipers at regular intervals. As described in Example 9,some mice are sacrificed for ex vivo tumor analysis using flowcytometry, while other mice are rechallenged to determine the impact ofthe memory response on tumor growth.

Example 15: A Mouse Prostate Cancer Model

A Veillonella strain is tested for its efficacy in the mouse model ofprostate cancer, either alone or in combination with other cancertherapies, including checkpoint inhibitor(s). Mice are divided intogroups receiving Veillonella Strain A, Veillonella Strain B, VeillonellaStrain C, and/or other Veillonella strain, with or without checkpointinhibitor treatment. As described in Example 9, Veillonella isadministered at varied doses at defined intervals. For example, somemice receive A bacterial strain (p.o.) on the day following tumor cellinjection (day 1). Some mice receive seven (7) consecutive doses ofVeillonella (one dose per day on days 14-21). Other mice receive dailydosing or, alternatively, some mice receive dosing every other day.Alternatively, mice are randomized into various treatment groups at adefined timepoint (e.g. on day 13) or when the tumors reach a certainsize (e.g. 100 mm³) and treatment is then initiated accordingly.

Mouse prostate cancer cells (1×10⁵ RM-1 cells or an appropriate numberof cells from another prostate cancer cell line) are injected intosyngeneic mice. Tumors from the various treatment groups are measuredwith calipers at regular intervals. As described in Example 9, some miceare sacrificed for ex vivo tumor analysis using flow cytometry, whileother mice are rechallenged to determine the impact of the memoryresponse on tumor growth.

Example 16: A Mouse Plasmacytoma Model

A bacterial strain is tested for its efficacy in the mouse model ofplasmacytoma, either alone or in combination with other cancertherapies, including checkpoint inhibitor(s). Mice are divided intogroups receiving Veillonella Strain A, Veillonella Strain B, VeillonellaStrain C, and/or other Veillonella strain, with or without checkpointinhibitor treatment. As described in Example 9, A bacterial strain isadministered at varied doses at defined intervals. For example, somemice receive A bacterial strain (p.o.) on the day following tumor cellinjection (day 1). Some mice receive seven (7) consecutive doses ofVeillonella (one dose per day on days 14-21). Other mice receive dailydosing or, alternatively, some mice receive dosing every other day.Alternatively, mice are randomized into various treatment groups at adefined timepoint (e.g. on day 13) or when the tumors reach a certainsize (e.g. 100 mm³) and treatment is then initiated accordingly.

Mineral Oil Induced Model of Plasmacytoma

To examine the efficacy of Veillonella in a plasmacytoma or multiplemyeloma model, mice are injected intraperitoneally three times with 500ul of 2,6,10,12-tetramethylpentadecane (“pristane oil”) at various timepoints between 0 and 60 days, as described by Potter et al. 1983.Peritoneal plasmacytomagenesis in mice: comparison of different pristanedose regimens. J. Natl. Cancer Inst. 71(2):391-5 (see also Lattanzio etal. 1997. Defective Development of Pristane-Oil Induced Plasmacytomas inInterleukin-6-Deficient BALB/C Mice. Am. J. Pathology: 151(3):689696).Progression of disease is measured by the degree of abdominal swellingand immune cells and particles in the ascites. Ascites fluid is analyzedfor immune cell phenotype by flow cytometry.

Cell-Line Induced Model of Plasmacytoma

To examine the efficacy of Veillonella in a plasmacytoma or multiplemyeloma model, either MOPC-104E cells or J558 plasmacytoma cells (TIB-6ATCC) are injected subcutaneously into one or more hind flanks of Balb/cmice (5×10⁶ cells), based on model described by Bhoopalam et al. 1980.Effect of dextran-S(alpha, 1-3 dextran) on the growth of plasmacytomasMOPC-104E and J558. J. Immunol. 125(4):1454-8 (see also Wang et al.2015. IL-10 enhances CTL-mediated tumor rejection by inhibiting highlysuppressive CD4+ T cells and promoting CTL persistence in a murine modelof plasmacytoma. OncoImmunology. 4(7): e1014232-1-9). Mice are dividedinto groups receiving Veillonella by oral gavage, and with or withoutcheckpoint inhibitor treatment. Tumors from the various treatment groupsare measured with calipers at regular intervals. As described in Example9, some mice are sacrificed for ex vivo tumor analysis using flowcytometry, while other mice are rechallenged to determine the impact ofthe memory response on tumor growth.

Example 17: A SCID Mouse Model of Mouse Myeloma

Veillonella is tested for its efficacy in the SCID mouse model ofmyeloma, either alone or in combination with other cancer therapies,including checkpoint inhibitor(s). Mice are divided into groupsreceiving Veillonella Strain A, Veillonella Strain B, Veillonella StrainC, and/or other Veillonella strain, with or without checkpoint inhibitortreatment. As described in Example 9, Veillonella is administered atvaried doses at defined intervals. For example, some mice receive Abacterial strain (p.o.) on the day following tumor cell injection (day1). Some mice receive seven (7) consecutive doses of Veillonella (onedose per day on days 14-21). Other mice receive daily dosing or,alternatively, some mice receive dosing every other day. Alternatively,mice are randomized into various treatment groups at a defined timepoint(e.g. on day 13) or when the tumors reach a certain size (e.g. 100 mm³)and treatment is then initiated accordingly.

To examine the efficacy of Veillonella using a human plasma cellleukemia, 1×10⁷ human myeloma cell lines, ARH77 cells (ARH77-ATCCCRL-1621, or an appropriate number of cells from another myeloma cellline such as KPMM2) are used. Myeloma cells are injected subcutaneouslyinto one or both hind flanks of SCID mice (See Caers et al. 2004. Ofmice and men: disease models of multiple myeloma. Drug Discovery Today:Disease Models. 1(4):373-380. Tumors from the various treatment groupsare measured with calipers at regular intervals. As described in Example9, some mice are sacrificed for ex vivo tumor analysis using flowcytometry, while other mice are rechallenged to determine the impact ofthe memory response on tumor growth.

Example 18: A Mouse Renal Cell Carcinoma Model

A bacterial strain is tested for its efficacy in the mouse model ofrenal cell carcinoma, either alone or in combination with other cancertherapies, including checkpoint inhibitor(s). Mice are divided intogroups receiving Veillonella Strain A, Veillonella Strain B, VeillonellaStrain C, and/or other Veillonella strain, with or without checkpointinhibitor treatment. As described in Example 9, Veillonella may beadministered at varied doses at defined intervals. For example, somemice receive A bacterial strain (p.o.) on the day following tumor cellinjection (day 1). Some mice receive seven (7) consecutive doses of aVeillonella bacterial strain (one dose per day on days 14-21). Othermice receive daily dosing or, alternatively, some mice receive dosingevery other day. Alternatively, mice are randomized into varioustreatment groups at a defined timepoint (e.g. on day 13) or when thetumors reach a certain size (e.g. 100 mm³) and treatment is theninitiated accordingly.

To examine the efficacy of Veillonella in a mouse model of renal cellcarcinoma, Renca cells (ATCC CRL-2947) or other renal cell carcinomacells are injected subcutaneously into one or both flanks of 7-8 weekold syngeneic Balb/c mice (5×10⁶ in 0.1 ml PBS). Tumors from the varioustreatment groups are measured with calipers at regular intervals. Asdescribed in Example 9, some mice are sacrificed for ex vivo tumoranalysis using flow cytometry, while other mice are rechallenged todetermine the impact of the memory response on tumor growth.

Example 19: A Mouse Bladder Cancer Model

A bacterial strain is tested for its efficacy in the mouse model ofbladder cancer, either alone or in combination with other cancertherapies, including checkpoint inhibitor(s). Mice are divided intogroups receiving Veillonella Strain A, Veillonella Strain B, VeillonellaStrain C, and/or other Veillonella strain, with or without checkpointinhibitor treatment. As described in Example 9, Veillonella may beadministered at varied doses at defined intervals. For example, somemice receive A bacterial strain (p.o.) on the day following tumor cellinjection (day 1). Some mice receive seven (7) consecutive doses of abacterial strain (one dose per day on days 14-21). Other mice receivedaily dosing or, alternatively, some mice receive dosing every otherday. Alternatively, mice are randomized into various treatment groups ata defined timepoint (e.g. on day 13) or when the tumors reach a certainsize (e.g. 100 mm³) and treatment is then initiated accordingly.

On the day of inoculation, MBT-2 cells (or other bladder cancer cellline) are harvested and resuspended in 1:1 PBS/Matrigel mixture. 2×10⁵MBT-2 cells are suspended in 100 ul of mixture and injectedsubcutaneously into one or both hind flanks of syngeneic mice. Tumorsare measured with calipers at regular intervals.

As described in Example 9, some mice are sacrificed for ex vivo tumoranalysis using flow cytometry, while other mice are rechallenged todetermine the impact of the memory response on tumor growth.

Example 20: A Mouse Model for Colorectal Carcinoma

A bacterial strain is tested for its efficacy in the mouse model ofcolorectal carcinoma, either alone or in combination with other cancertherapies, including checkpoint inhibitor(s). Mice are divided intogroups receiving Veillonella Strain A, Veillonella Strain B, VeillonellaStrain C, and/or other Veillonella strain, with or without checkpointinhibitor treatment. As described in Example 9, Veillonella may beadministered at varied doses at defined intervals. For example, somemice receive A bacterial strain (p.o.) on the day following tumor cellinjection (day 1). Some mice receive seven (7) consecutive doses of Abacterial strain (one dose per day on days 14-21). Other mice receivedaily dosing or, alternatively, some mice receive dosing every otherday. Alternatively, mice are randomized into various treatment groups ata defined timepoint (e.g. on day 13) or when the tumors reach a certainsize (e.g. 100 mm³) and treatment is then initiated accordingly.

Female 6-8 week old Balb/c mice are obtained from Taconic (Germantown,N.Y.). 100,000 CT-26 colorectal tumor cells (ATCC CRL-2638) wereresuspended in sterile PBS and inoculated in the presence of 50%Matrigel. CT-26 tumor cells were subcutaneously injected into one hindflank of each mouse. Treatment with a Veillonella strain is initiated atsome point following tumor cell inoculation at varied doses and atdefined intervals. For example, some mice receive between1-5×10{circumflex over ( )}9 CFU (100 μl final volume) per dose.Possible routes of administration include oral gavage (p.o.),intravenous injection, intratumoral injection (IT) or peritumoral orsubtumoral or subcutaneous injection. In order to assess the systemicanti-tumoral effects of Veillonella treatment, additional mice may beinoculated with tumor cells in the contralateral (untreated, second)flank prior to IT, peritumoral, or subtumoral treatment with Veillonellain the first flank.

Tumors from the various treatment groups are measured with calipers atregular intervals. As described in Example 9, some mice are sacrificedfor ex vivo tumor analysis using flow cytometry, while other mice arerechallenged to determine the impact of the memory response on tumorgrowth.

Example 21: Manufacturing Conditions

Enriched media is used to grow and prepare the bacterium for in vitroand in vivo use. For example, media may contain sugar, yeast extracts,plant based peptones, buffers, salts, trace elements, surfactants,anti-foaming agents, and vitamins. Composition of complex componentssuch as yeast extracts and peptones may be undefined or partiallydefined (including approximate concentrations of amino acids, sugarsetc.). Microbial metabolism may be dependent on the availability ofresources such as carbon and nitrogen. Various sugars or other carbonsources may be tested. Alternatively, media may be prepared and theselected bacterium grown as shown by Saarela et al., J. AppliedMicrobiology. 2005. 99: 1330-1339, which is hereby incorporated byreference. Influence of fermentation time, cryoprotectant andneutralization of cell concentrate on freeze-drying survival, storagestability, and acid and bile exposure of the selected bacterium producedwithout milk-based ingredients.

At large scale, the media is sterilized. Sterilization may be by UltraHigh Temperature (UHT) processing. The UHT processing is performed atvery high temperature for short periods of time. The UHT range may befrom 135-180° C. For example, the medium may be sterilized from between10 to 30 seconds at 135° C.

Inoculum can be prepared in flasks or in smaller bioreactors and growthis monitored. For example, the inoculum size may be betweenapproximately 0.5 and 3% of the total bioreactor volume. Depending onthe application and need for material, bioreactor volume can be at least2 L, 10 L, 80 L, 100 L, 250 L, 1000 L, 2500 L, 5000 L, 10,000 L.

Before the inoculation, the bioreactor is prepared with medium atdesired pH, temperature, and oxygen concentration. The initial pH of theculture medium may be different that the process set-point. pH stressmay be detrimental at low cell centration; the initial pH could bebetween pH 7.5 and the process set-point. For example, pH may be setbetween 4.5 and 8.0. During the fermentation, the pH can be controlledthrough the use of sodium hydroxide, potassium hydroxide, or ammoniumhydroxide. The temperature may be controlled from 25° C. to 45° C., forexample at 37° C. Anaerobic conditions are created by reducing the levelof oxygen in the culture broth from around 8 mg/L to 0 mg/L. Forexample, nitrogen or gas mixtures (N2, CO2, and H2) may be used in orderto establish anaerobic conditions. Alternatively, no gases are used andanaerobic conditions are established by cells consuming remaining oxygenfrom the medium. Depending on strain and inoculum size, the bioreactorfermentation time can vary. For example, fermentation time can vary fromapproximately 5 hours to 48 hours.

For example, a frozen vial is diluted to 0.1% in a 1 L media at 37 C for12-16 hours. The media is PM11 with 1 g/l L-sodium lactate (no FeSO4, noNH4Cl, no malate). The 1 L media is diluted to 1% in a 15 L bioreactorat 37, 150 rpm, gas of 5% CO2 and 95% N2, uncontrolled pH for 16-18hours. The feed is 10×YEP, 33 g/l L-sodium lactate (no G2) (Constantfeed: 11 mL/Lh). It is then centrifuged at 10,000 g, 10C, for 10 minutesto collect 90 g pellet/15 L. Then placed in a new stabilizer:sucrose-dextran-cycteine 0.18 g stab/g pellet.

Reviving microbes from a frozen state may require specialconsiderations. Production medium may stress cells after a thaw; aspecific thaw medium may be required to consistently start a seed trainfrom thawed material. The kinetics of transfer or passage of seedmaterial to fresh medium, for the purposes of increasing the seed volumeor maintaining the microbial growth state, may be influenced by thecurrent state of the microbes (ex. exponential growth, stationarygrowth, unstressed, stressed).

Inoculation of the production fermenter(s) can impact growth kineticsand cellular activity. The initial state of the bioreactor system mustbe optimized to facilitate successful and consistent production. Thefraction of seed culture to total medium (e.g. a percentage) has adramatic impact on growth kinetics. The range may be 1-5% of thefermenter's working volume. The initial pH of the culture medium may bedifferent from the process set-point. pH stress may be detrimental atlow cell concentration; the initial pH may be between pH 7.5 and theprocess set-point. Agitation and gas flow into the system duringinoculation may be different from the process set-points. Physical andchemical stresses due to both conditions may be detrimental at low cellconcentration.

Process conditions and control settings may influence the kinetics ofmicrobial growth and cellular activity. Shifts in process conditions maychange membrane composition, production of metabolites, growth rate,cellular stress, etc. Optimal temperature range for growth may vary withstrain. The range may be 20-40° C. Optimal pH for cell growth andperformance of downstream activity may vary with strain. The range maybe pH 5-8. Gasses dissolved in the medium may be used by cells formetabolism. Adjusting concentrations of O₂, CO₂, and N₂ throughout theprocess may be required. Availability of nutrients may shift cellulargrowth. Microbes may have alternate kinetics when excess nutrients areavailable.

The state of microbes at the end of a fermentation and during harvestingmay impact cell survival and activity. Microbes may be preconditionedshortly before harvest to better prepare them for the physical andchemical stresses involved in separation and downstream processing. Achange in temperature (often reducing to 20-5° C.) may reduce cellularmetabolism, slowing growth (and/or death) and physiological change whenremoved from the fermenter. Effectiveness of centrifugal concentrationmay be influenced by culture pH. Raising pH by 1-2 points can improveeffectiveness of concentration but can also be detrimental to cells.Microbes may be stressed shortly before harvest by increasing theconcentration of salts and/or sugars in the medium. Cells stressed inthis way may better survive freezing and lyophilization duringdownstream.

Separation methods and technology may impact how efficiently microbesare separated from the culture medium. Solids may be removed usingcentrifugation techniques. Effectiveness of centrifugal concentrationcan be influenced by culture pH or by the use of flocculating agents.Raising pH by 1-2 points may improve effectiveness of concentration butcan also be detrimental to cells. Microbes may be stressed shortlybefore harvest by increasing the concentration of salts and/or sugars inthe medium. Cells stressed in this way may better survive freezing andlyophilization during downstream. Additionally, Microbes may also beseparated via filtration. Filtration is superior to centrifugationtechniques for purification if the cells require excessive g-minutes tosuccessfully centrifuge. Excipients can be added before afterseparation. Excipients can be added for cryo protection or forprotection during lyophilization. Excipients can include, but are notlimited to, sucrose, trehalose, or lactose, and these may bealternatively mixed with buffer and anti-oxidants. Prior tolyophilization, droplets of cell pellets mixed with excipients aresubmerged in liquid nitrogen.

Harvesting can be performed by continuous centrifugation. Product may beresuspended with various excipients to a desired final concentration.Excipients can be added for cryo protection or for protection duringlyophilization. Excipients can include, but are not limited to, sucrose,trehalose, or lactose, and these may be alternatively mixed with bufferand anti-oxidants. Prior to lyophilization, droplets of cell pelletsmixed with excipients are submerged in liquid nitrogen.

Lyophilization of material, including live bacteria, begins with primarydrying. During the primary drying phase, the ice is removed. Here, avacuum is generated and an appropriate amount of heat is supplied to thematerial for the ice to sublime. During the secondary drying phase,product bound water molecules are removed. Here, the temperature israised higher than in the primary drying phase to break anyphysico-chemical interactions that have formed between the watermolecules and the product material. The pressure may also be loweredfurther to enhance desorption during this stage. After the freeze-dryingprocess is complete, the chamber may be filled with an inert gas, suchas nitrogen. The product may be sealed within the freeze dryer under dryconditions, preventing exposure to atmospheric water and contaminants.

Example 22: Intravenously Administered Veillonella Inhibits ColorectalCarcinoma Tumor Growth

Female 6-8 week old Balb/c mice were obtained from Taconic (Germantown,N.Y.). 100,000 CT-26 colorectal tumor cells (ATCC CRL-2638) wereresuspended in sterile PBS and inoculated in the presence of 50%Matrigel. CT-26 tumor cells were subcutaneously injected into one hindflank of each mouse. When tumor volumes reached an average of 100 mm³(approximately 10-12 days following tumor cell inoculation), animalswere distributed into the following groups: 1) Vehicle; 2) VeillonellaStrain A; 3) Veillonella Strain B and 4) anti-PD-1 antibody. Antibodieswere administered intraperitoneally (i.p.) at 200 μg/mouse (100 μl finalvolume) every four days, starting on day 1, for a total of 3 times(Q4Dx3) and Veillonella EVs (5 μg) were intravenously (i.v.) injectedevery third day, starting on day 1 for a total of 4 times (Q3Dx4). BothVeillonella groups showed tumor growth inhibition greater than that seenin the anti-PD-1 group (FIGS. 4 and 5). EVs were prepared and quantifiedper Example 27.

Example 23: Efficacy of EVs Varies Based on Source Microbe, Dose, andRoute of Administration

Female 6-8 week old Balb/c mice were obtained from Taconic (Germantown,N.Y.). 100,000 CT-26 colorectal tumor cells (ATCC CRL-2638) wereresuspended in sterile PBS and inoculated in the presence of 50%Matrigel. CT-26 tumor cells were subcutaneously injected into one hindflank of each mouse. When tumor volumes reached an average of 100 mm³(approximately 10-12 days following tumor cell inoculation), animalswere distributed into the following groups as highlighted in Table 2.

TABLE 2 Treatment Groups Dose/Route/ Group Treatment Schedule 1 IVVehicle (PBS) N/A/IV/Q3Dx4 2 PO Vehicle (sucrose) N/A/PO/QD 3 Anti-PD-1200 μg/IP/Q4Dx3 4 Veillonella parvula  10 μg/IV/Q3Dx4 Strain B EV 5Veillonella parvula  5 μg/IV/Q3Dx4 Strain B EV 6 Veillonella parvula  2μg/IV/Q3Dx4 Strain B EV 7 Veillonella tobetsuensis 75 μg/PO. QD Strain AEV 8 Veillonella tobetsuensis  5 μg/IV/Q3Dx4 Strain A EV

As noted in the table, antibodies were administered intraperitoneally(i.p.) at 200 μg/mouse (100 μl final volume) every four days, startingon day 1, for a total of 3 times (Q4Dx3) and EVs when administeredintravenously (i.v.) were injected every third day, starting on day 1for a total of 4 times (Q3Dx4). The treatment groups administered bymouth (p.o.) were administered daily (QD). Efficacy of Veillonella EVsvaries based on source microbe, dose, and route of administration (FIGS.6 and 7).

Example 24: Immune Modulation of Human Commensal Bacteria in a KLH-BasedDelayed Type Hypersensitivity Model

Similar to Example 1, mice were sensitized to KLH as described above,and groups received live or irradiated Veillonella (25 kGy). Micereceived vehicle, Dexamethasone, irradiated Veillonella Strain D(8.32×10{circumflex over ( )}9, 25 kGy), irradiated Veillonella Strain E(3.28×10{circumflex over ( )}9, 25 kGy), irradiated Veillonella Strain F(5.38×10{circumflex over ( )}9, 25 kGy), or irradiated VeillonellaStrain G (2.01×10{circumflex over ( )}9, 25 kGy). Mice were dosed ondays 1-9, and challenged on day 8 with ear measurements taken on day 9(24 hours) and day 10 (48 hours).

As shown in FIG. 8, irradiated Veillonella Strains are efficacious inreducing ear swelling at 24 hours compared to vehicle (negativecontrol). As shown in FIG. 9, the results of Veillonella Strains B, E,F, and G reducing antigen-specific ear inflammation at 24 hours comparedto vehicle (negative control) and anti-inflammatory Dexamethasone(positive control) following antigen challenge in a KLH-based delayedtype hypersensitivity model. Both live and irradiated Veillonella StrainB and E were efficacious at inhibiting ear inflammation, but irradiatedVeillonella Strain E was even more efficacious than live Strain E. ForVeillonella Strain F, irradiation gamma-irradiation caused anon-performing strain of Veillonella to become efficacious. All thegroups received 10 mg of the powder per dose.

Example 25: Generation of Veillonella LPS Mutant

Antibiotic resistance can be classified as intrinsic, acquired, oradaptive. Adaptive resistance is defined as reduced antimicrobialkilling in populations of bacteria that were originally susceptible to aparticular antibiotic agent. It involves a transient increase in theability of bacteria to survive the antibiotic, mainly because ofalterations in gene and/or protein expression levels triggered byenvironmental conditions such as stress, nutrient conditions, andsub-inhibitory levels of the antibiotic. In contrast to intrinsic andacquired resistance mechanisms, which are stable and can be transmittedto progeny, adaptive resistance is transient and is usually lost uponremoval of the antibiotic agent. This type of resistance has beenreported for aminoglycosides and polymyxins (polymyxin B and colistin)in bacteria, especially Gram-negative.

Adaptive resistance might be one of the reasons of the phenomenon thatlaboratory susceptibility results are not congruent with the clinicaleffectiveness of antibiotics. Polymyxin resistance in bacteria is knownto be adaptive, which is characterized by induction of resistance in thepresence of drug and reversal to the susceptible phenotype in itsabsence. Polymyxins bind to lipopolysaccharide (LPS), the majorconstituent of the outer membrane in Gram-negative bacteria, throughinteractions with phosphates and fatty acids of LPS core and lipid Amoieties. These interactions subsequently result in cell lysis anddeath.

Polymyxin resistance in Gram-negative bacteria is associated with theaddition of 4-amino-L-arabinose (L-Ara4N) or phosphoethanolamine (pEtN)to lipid A and core oligosaccharide components. This results in areduction of the net negative charge of the outer membrane. Theregulatory two-component systems (TCSs) PhoP-PhoQ (PhoPQ) and PmrA-PmrB(PmrAB) play important roles in lipid A modification, which subsequentlyresults in bacteria becoming resistant to polymyxins. Further, thesurvival rates of bacteria comprising deletion mutants (lpxC and/orpmrB), which are involved in LPS biosynthesis and modification, weredecreased >4-fold when colistin was present at sub-inhibitoryconcentrations, compared with those for their WT parents.

The evolution of colistin resistance is due to the modification of lipidA in LPS. This results in a reduction of the net charge of the outermembrane. This modification is known to be regulated by severaltwo-component systems (TCSs), including PhoPQ, PmrAB, ParRS, and CprRS.Loss of LPS due to mutations or disruption to genes involved in lipid Abiosynthesis, such as lpxA, lpxC, and lpxD, has been reported.

Veillonella LPS mutants are generated by serial passages in the presenceof colistin using methods known to those skilled in the art (J Y Lee etal. Sci Rep. 2016 May 6; 6:25543).

For example, Veillonella cultures are routinely grown on BRU agar plates(Anaerobic Systems) at 37 C for 2-4 days. Veillonella cultures are grownin liquid medium in the presence of a range of colistin concentration(0.1 to 16 mg/L) for 1 to 3 days in order to determine minimuminhibitory concentration of colistin. Veillonella cultures are grown inliquid medium in the presence of sub-inhibitory colistin concentration(i.e. less than minimum inhibitory concentration) for 1 to 3 days toallow bacterial growth. Grown cultures are diluted 25- to 50-fold infresh medium containing 2-fold higher concentration of colistin. Eachbacterial culture media contained 0.13, 0.25, 0.5, 1, 2, 4, 8, 16, 32,64, 128, or 256 mg/L colistin as bacterial cultures serially passagedfor 10-14 generations. When bacterial cultures are unable to grow inliquid medium with 2-fold higher concentration of colistin, cultures areplated on BRU agar plates and allowed to grow for 2-4 days. Bacterialcolonies are scooped from the plates to start cultures in liquid mediumcontaining colistin at a concentration that allowed bacterial growth.Serial passages are repeated until bacteria are resistant to colistinat >250 mg/L concentration. Following the last passage, bacterialcolonies are grown on BRU plates containing 100 mg/L colistin andseveral individual colonies are selected for lipopolysaccharide (LPS)analysis.

Colistin-resistant Veillonella clones are selected for LPS analysis.Analytical methods include SDS-PAGE analysis followed by ProQ LPSstaining (Invitrogen), chromogenic LAL endotoxin assay (GeneScript),lipid A analysis by MALDI-TOF MS analysis (Bruker).

Example 26: Preparation and Purification of EVs from Bacteria

Extracellular vesicles (EVs) are prepared from bacterial cultures usingmethods known to those skilled in the art (S. Bin Park, et al. PLoS ONE.6(3):e17629 (2011)).

For example, bacterial cultures are centrifuged at 11,000×g for 20-40min at 4° C. to pellet bacteria. Culture supernatants are then passedthrough a 0.22 μm filter to exclude intact bacterial cells. Filteredsupernatants are concentrated using methods that may include, but arenot limited to, ammonium sulfate precipitation, ultracentrifugation, orfiltration. Briefly, for ammonium sulfate precipitation, 1.5-3 Mammonium sulfate is added to filtered supernatant slowly, while stirringat 4° C. Precipitations are incubated at 4° C. for 8-48 hours and thencentrifuged at 11,000×g for 20-40 min at 4° C. The pellets containbacterial EVs and other debris. Briefly, using ultracentrifugation,filtered supernatants are centrifuged at 100,000-200,000×g for 1-16hours at 4° C. The pellet of this centrifugation contains bacterial EVsand other debris. Briefly, using a filtration technique, using an AmiconUltra spin filter or by tangential flow filtration, supernatants arefiltered so as to retain species of molecular weight >50 or 100 kDa.

Alternatively, EVs are obtained from bacterial cultures continuouslyduring growth, or at selected time points during growth, by connecting abioreactor to an alternating tangential flow (ATF) system (e.g., XCellATF from Repligen) according to manufacturer's instructions. The ATFsystem retains intact cells (>0.22 um) in the bioreactor, and allowssmaller components (e.g., EVs, free proteins) to pass through a filterfor collection. For example, the system may be configured so that the<0.22 um filtrate is then passed through a second filter of 100 kDa,allowing species such as EVs between 0.22 um and 100 kDa to becollected, and species smaller than 100 kDa to be pumped back into thebioreactor. Alternatively, the system may be configured to allow formedium in the bioreactor to be replenished and/or modified during growthof the culture. EVs collected by this method may be further purifiedand/or concentrated by ultracentrifugation or filtration as describedabove for filtered supernatants.

EVs obtained by methods described above may be further purified bygradient ultracentrifugation, using methods that may include, but arenot limited to, use of a sucrose gradient or Optiprep gradient. Briefly,using a sucrose gradient method, if ammonium sulfate precipitation orultracentrifugation were used to concentrate the filtered supernatants,pellets are resuspended in 60% sucrose, 30 mM Tris, pH 8.0. Iffiltration was used to concentrate the filtered supernatant, theconcentrate is buffer exchanged into 60% sucrose, 30 mM Tris, pH 8.0,using an Amicon Ultra column. Samples are applied to a 35-60%discontinuous sucrose gradient and centrifuged at 200,000×g for 3-24hours at 4° C. Briefly, using an Optiprep gradient method, if ammoniumsulfate precipitation or ultracentrifugation were used to concentratethe filtered supernatants, pellets are resuspended in 35% Optiprep inPBS. If filtration was used to concentrate the filtered supernatant, theconcentrate is diluted using 60% Optiprep to a final concentration of35% Optiprep. Samples are applied to a 35-60% discontinuous sucrosegradient and centrifuged at 200,000×g for 3-24 hours at 4° C.

To confirm sterility and isolation of the EV preparations, EVs areserially diluted onto agar medium used for routine culture of thebacteria being tested, and incubated using routine conditions.Non-sterile preparations are passed through a 0.22 um filter to excludeintact cells. To further increase purity, isolated EVs may be DNase orproteinase K treated.

Alternatively, for preparation of EVs used for in vivo injections,purified EVs are processed as described previously (G. Norheim, et al.PLoS ONE. 10(9): e0134353 (2015)). Briefly, after sucrose gradientcentrifugation, bands containing EVs are resuspended to a finalconcentration of 50 μg/mL in a solution containing 3% sucrose or othersolution suitable for in vivo injection known to one skilled in the art.This solution may also contain adjuvant, for example aluminum hydroxideat a concentration of 0-0.5% (w/v).

To make samples compatible with further testing (e.g. to remove sucroseprior to TEM imaging or in vitro assays), samples are buffer exchangedinto PBS or 30 mM Tris, pH 8.0 using filtration (e.g. Amicon Ultracolumns), dialysis, or ultracentrifugation (200,000×g, ≥3 hours, 4° C.)and resuspension.

Example 27: Preparation and Purification of Veillonella EVs fromBacteria Prep Method:

Bacterial cultures were centrifuged at 10,000-16,000×g for 10-15 min at4° C., 10° C., or room temperature to pellet bacteria. Culturesupernatants were then filtered to ≤0.22 μm to exclude intact bacterialcells. Filtered supernatants were concentrated and buffer exchanged intoPBS by tangential flow filtration, retaining species >100 kDa.Supernatants were then filtered again to ≤0.22 μm and EVs were pelletedby ultracentrifugation at 200,000×g for 1 h at 4° C. Pellets wereresuspended in PBS and further purified by gradient ultracentrifugation.Samples were diluted to 45% Optiprep with 3 volumes of 60% Optiprep andapplied to the bottom of a 0-45% discontinuous Optiprep gradient.Gradients were centrifuged at 200,000×g for 4-24 hours at 4° C.EV-containing fractions from above the level of the original sample wereremoved, diluted at least 15-fold with PBS and pelleted byultracentrifugation at 200,000×g for 1 h at 4° C. Pellets wereresuspended in PBS. Prior to in vivo administration, samples weresterile filtered to ≤0.22 μm.

Quantification:

Dosing of EVs was based on particle counts, as assessed by NanoparticleTracking Analysis (NTA) using a NanoSight NS300 (Malvern Panalytical)according to manufacturer instructions. Counts for each sample werebased on at least three videos of 30 sec duration each, counting 40-140particles per frame, with a syringe pump speed of 75. Protein amountswere also tracked and quantifed per dose for each EV prep. Total proteinwas quantified by Bradford assays using Quick Start Bradford 1× dyereagent (Bio-Rad) according to manufacturer instructions.

Example 28: Preparation of Veillonella Bacteria Feed Preparation forFed-Batch Process

Components Per 3 L (g) Yeast Extract 19512 Organotechnie S.A.S. 300 SoyPeptone E110 19885 Organotechnie S.A.S. 300 Potato Peptone E210 19425Organotechnie S.A.S. 150 Soy Peptone A3 SC 19685 Organotechnie S.A.S.300

3 L of DI water is heated in microwave for about 6 min (at 50-60C). The2L and 3L levels in 5 L beaker are marked. 2 L DI water is added intothe beaker, with the largest magnet. The heater and stirrer (200 rpm)are turned on. The solution's temperature is kept between 50-60C duringthe mixing. The components in the above table are added one by one. 100g maximum is added each time, until it is completely dissolved beforeadding the following component. After adding the last component,dissolve it completely for at least 30 min. Final volume is about 2800ml. The solution is filtered sterilized. 1 L sterile bottles (945 ml ofthe solution to in 1 L bottle ×3) is used. 55 ml of 60% L-sodium lactateis added (to get 33 g/l lactate in the feed) to each bottle aseptically.

L-sodium lactate stocks are prepared and sterilized in advance.

Inoculum Preparation from Frozen Stock:

PM11 (with 5 g/l sodium-L-lactate) is prepared in advance following thePM11 media preparation protocol. The media is transferred to the coy anddegassed overnight before inoculating. The frozen vial is transferred tothe coy. Its cap is cleaned with ethanol wipe. After the vial thawed, itis vortexed gently and 1 ml of the stock is transferred to the 1 L PM11media immediately (0.1% inoculum). The media is incubated at 37 Covernight. The quality of the inoculum determines the incubation time.Therefore, at least in first couple of runs, it is recommended to followthe growth to get an idea on inoculum stage. The harvest point is verycrucial for the inoculum. The inoculum should be at mid or lateexponential phase.

PM11 Media (Filter Sterilization)

PM11 g/L Sodium L-Lactate 5 Yeast Extract 19512 Organotechnie S.A.S. 10Soy Peptone E110 19885 Organotechnie S.A.S. 10 Potato Peptone E210 19425Organotechnie S.A.S. 5 Soy Peptone A3 SC 19685 Organotechnie S.A.S. 10Dipotassium Phosphate K2HPO4 5.03 Monopotassium Phosphate KH2PO4 2.87L-Cysteine-HCl 0.5 Tri-sodium citrate 5 Magnesium chloride 0.5 Manganesechloride 0.1

Group 1 components g for L Yeast Extract 19512 Organotechnie S.A.S. 10Soy Peptone E110 19885 Organotechnie S.A.S 10 Soy Peptone A3 SC 19685Organotechnie S.A.S 10 Potato Peptone E210 19425 Organotechnie S.A.S 5Dipotassium Phosphate K2HPO4 5.03 Monopotassium Phosphate KH2PO4 2.87Tri-sodium citrate 5 Sodium L-Lactate 5

Medium Preparation:

2 L beaker is placed on a scale and tare and filled with 800 g of water.A magnet is placed and stirring begins. Each of the Group 1 componentsexcept Sodium L-Lactate is weight out. Each component, except for theSodium L-Lactate, is added one by one to the mixing vessel, making surethat each component is totally dissolved before adding the nextcomponent. Once all solids have completely dissolved, the stirrer isstopped. QS to a mass of 1 kg with DI water. The mix is filtersterilized with a 0.2 um sterilizing grade filter. 500 mL of SodiumL-Lactate acid is autoclave sterilized at 121 C for 30 minutes. 10 mL ofthe Group 2 solution is added to the group and mixed aseptically. 8.3 mLof sterilized Sodium L-Lactate is added to the mix aseptically. Themedia is labeled and transferred to the coy. Loosen its cap and let itdegas overnight.

Group 2 components g for 1 L L-Cysteine-HCl 50 Magnesium chloride 50Manganese chloride 10

Group 2 Preparation:

About 700 ml is added into 1 L beaker and placed on hot plate and heatedto 50 C. Each of the Group 2 components is weight out. Components areadded one by one making sure that each component is totally dissolvedbefore adding the following component. Once everything is dissolved,using 1 L glass cylinder, QS to 1 L and filter-sterilized with 0.2 μmfilter. Label with name and date of preparation. Medium bottle iswrapped in aluminum foil and transferred to a COY, leave for max 2months.

Example 29: Intravenously Administered Veillonella EVs InhibitColorectal Carcinoma Tumor Growth

Female 6-8 week old Balb/c mice were obtained from Taconic (Germantown,N.Y.). 100,000 CT-26 colorectal tumor cells (ATCC CRL-2638) wereresuspended in sterile PBS and inoculated in the presence of 50%Matrigel. CT-26 tumor cells were subcutaneously injected into one hindflank of each mouse. When tumor volumes reached an average of 100 mm³(approximately 10-12 days following tumor cell inoculation), animalswere distributed into the following groups: 1) Vehicle; 2) anti-PD-1antibody; 3) V. parvula Strain A EVs 7.0e+10 particles, 4) V. atypicaStrain A EVs 2.0e+11 particles, 5) V. atypica Strain A EVs 7.0e+10particles, and 5) V. atypica Strain B EVs 1.5e+10 particles. Antibodieswere administered intraperitoneally (i.p.) at 200 ug/mouse (100 ul finalvolume) every four days, starting on day 1(IP Q4Dx3), and VeillonellaEVs (7.0e+10, 2.0e+11, or 1.5e+10 particles) were administered byintravenously (IV) daily (IV Q3Dx4), starting on day 1 until theconclusion of the study. The Veillonella group showed tumor growthinhibition comparable or more significant to that seen in the anti-PD-1group (FIGS. 14 and 15). EVs were prepared and quantified per Example27.

Dose Dose Gram (Particle (ug Route, Group Treatment Stain count)protein) Schedule 1 Vehicle (PBS) N.A. N.A. — IV Q3Dx4 (n = 10) 2Anti-PD-1 N.A. 200 ug — IP Q4Dx3 (n = 10) 3 EEV V. parvula — 7.0e+101.41 IV Q3Dx4 (n = 10) Strain A particles 7.0e+10PC (with lower totalprotein) 4 EEV V. atypica — 2.0e+11 9.94 IV Q3Dx4 (n = 8)  Strain Aparticles 2.0e+11PC 5 EEV V. atypica — 7.0e+10 3.48 IV Q3Dx4 (n = 8) Strain A particles 7.0e+10PC 6 EEV V. atypica — 1.5e+10 5.00 IV Q3Dx4 (n= 10) Strain B particles 1.5e+10PC

INCORPORATION BY REFERENCE

All publications patent applications mentioned herein are herebyincorporated by reference in their entirety as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference. In case of conflict, thepresent application, including any definitions herein, will control.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is: 1-161. (canceled)
 162. A method of treating animmune disorder in a subject comprising orally administering to thesubject a pharmaceutical composition comprising Veillonella parvulaStrain B (ATCC Accession Number PTA-125691) and a pharmaceuticallyacceptable carrier.
 163. The method of claim 162, wherein theVeillonella parvula Strain B (ATCC Accession Number PTA-125691) and thepharmaceutically acceptable carrier are within an enteric coating. 164.The method of claim 162, wherein the immune disorder is atopicdermatitis, psoriasis, or asthma.
 165. The method of claim 162, whereinat least 50% of the bacteria in the pharmaceutical composition areVeillonella parvula Strain B (ATCC Accession Number PTA-125691). 166.The method of claim 162, wherein at least 95% of the bacteria in thepharmaceutical composition are Veillonella parvula Strain B (ATCCAccession Number PTA-125691).
 167. The method of claim 162, wherein atleast 99% of the bacteria in the pharmaceutical composition areVeillonella parvula Strain B (ATCC Accession Number PTA-125691). 168.The method of claim 162, wherein the pharmaceutical compositioncomprises at least 1×10⁶ colony forming units (CFUs) of Veillonellaparvula Strain B (ATCC Accession Number PTA-125691.
 169. The method ofclaim 162, wherein the pharmaceutical composition comprises livebacteria.
 170. The method of claim 162, wherein the pharmaceuticalcomposition comprises killed bacteria.
 171. The method of claim 162,wherein the pharmaceutical composition comprises irradiated bacteria.172. The method of claim 171, wherein the irradiated bacteria comprisesgamma irradiated bacteria.
 173. The method of claim 163, wherein thepharmaceutical composition is formulated as an enteric coated capsule.174. The method of claim 163, wherein the pharmaceutical composition isformulated as an enteric coated tablet.
 175. The method of claim 163,wherein the enteric coating is a coating for duodenal release at a pH ofabout 5.5-6.2.
 176. The method of claim 163, wherein the enteric coatingis a coating for release in the ileum at a pH of about 7.2-7.2.
 177. Themethod of claim 163, wherein the enteric coating is a coating forrelease in the colon at a pH of about 5.6-7.2.
 178. The method of claim162, wherein the Veillonella parvula Strain B (ATCC Accession NumberPTA-125691) is freeze-dried.
 179. The method of claim 62, wherein themethod further comprises administering to the subject an additionaltherapeutic.
 180. The method of claim 179, wherein the additionaltherapeutic is an antibiotic.
 181. The method of claim 179, wherein theadditional therapeutic is an anti-inflammatory agent.